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What is a significant cause of Influenze like illness among healthy adolescents and adults presenting for medical evaluation?
|
HCoV
|
[
"Human coronavirus HCoV is a known cause of influenza‐like illness ILI . In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 34% , HCoV‐229E 28% , HCoV‐NL63 22% , and HCoV‐HKU1 16% . We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%.",
"Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%. By contrast, other HCoV species HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E are much more prevalent, much less severe, and common causes of influenza-like illness ILI . Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection.",
"6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded.",
"Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 .",
"Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus HCoV is an important cause of influenza-like illness ILI ranged from 4% to 22%. 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known.",
"8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation.",
"Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease."
] | 1,545 | 1,658 |
What is the most common species of Human Coronavirus among adults?
|
HCoV-OC43
|
[
"Human coronavirus HCoV is a known cause of influenza‐like illness ILI . In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 34% , HCoV‐229E 28% , HCoV‐NL63 22% , and HCoV‐HKU1 16% . We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%.",
"Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%. By contrast, other HCoV species HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E are much more prevalent, much less severe, and common causes of influenza-like illness ILI . Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection.",
"6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded.",
"Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 .",
"Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus HCoV is an important cause of influenza-like illness ILI ranged from 4% to 22%. 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known.",
"8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation.",
"Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease."
] | 1,545 | 1,659 |
Which Human Coronavirus showed species specific clinical characteristics of its infection?
|
HCoV‐HKU1
|
[
"Human coronavirus HCoV is a known cause of influenza‐like illness ILI . In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 34% , HCoV‐229E 28% , HCoV‐NL63 22% , and HCoV‐HKU1 16% . We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%.",
"Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%. By contrast, other HCoV species HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E are much more prevalent, much less severe, and common causes of influenza-like illness ILI . Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection.",
"6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded.",
"Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 .",
"Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus HCoV is an important cause of influenza-like illness ILI ranged from 4% to 22%. 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known.",
"8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation.",
"Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease."
] | 1,545 | 1,660 |
What causes the outbreak of SARS and MERS.
|
Highly virulent species of HCoV
|
[
"Human coronavirus HCoV is a known cause of influenza‐like illness ILI . In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 34% , HCoV‐229E 28% , HCoV‐NL63 22% , and HCoV‐HKU1 16% . We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%.",
"Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%. By contrast, other HCoV species HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E are much more prevalent, much less severe, and common causes of influenza-like illness ILI . Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection.",
"6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded.",
"Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 .",
"Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus HCoV is an important cause of influenza-like illness ILI ranged from 4% to 22%. 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known.",
"8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation.",
"Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease."
] | 1,545 | 1,717 |
What is the case fatality rate of SARS and MERS?
|
ranged from 14% to 45%
|
[
"Human coronavirus HCoV is a known cause of influenza‐like illness ILI . In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 34% , HCoV‐229E 28% , HCoV‐NL63 22% , and HCoV‐HKU1 16% . We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%.",
"Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%. By contrast, other HCoV species HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E are much more prevalent, much less severe, and common causes of influenza-like illness ILI . Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection.",
"6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded.",
"Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 .",
"Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus HCoV is an important cause of influenza-like illness ILI ranged from 4% to 22%. 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known.",
"8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation.",
"Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease."
] | 1,545 | 1,718 |
What were the common HCOV strains in the 5 year USA study?
|
HCoV-OC43 and HCoV-229E
|
[
"Human coronavirus HCoV is a known cause of influenza‐like illness ILI . In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 34% , HCoV‐229E 28% , HCoV‐NL63 22% , and HCoV‐HKU1 16% . We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%.",
"Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%. By contrast, other HCoV species HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E are much more prevalent, much less severe, and common causes of influenza-like illness ILI . Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection.",
"6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded.",
"Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 .",
"Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus HCoV is an important cause of influenza-like illness ILI ranged from 4% to 22%. 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known.",
"8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation.",
"Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease."
] | 1,545 | 1,719 |
Which species are more prevalent but less severe?
|
HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E
|
[
"Human coronavirus HCoV is a known cause of influenza‐like illness ILI . In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 34% , HCoV‐229E 28% , HCoV‐NL63 22% , and HCoV‐HKU1 16% . We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%.",
"Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%. By contrast, other HCoV species HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E are much more prevalent, much less severe, and common causes of influenza-like illness ILI . Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection.",
"6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded.",
"Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 .",
"Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus HCoV is an important cause of influenza-like illness ILI ranged from 4% to 22%. 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known.",
"8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation.",
"Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease."
] | 1,545 | 1,720 |
What kinds of viruses are Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV)?
|
arboviruses
|
[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553 | 3,003 |
What are the current clinically-available methods to detect encephalitis viral antigens?
|
ELISA and IFA
|
[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553 | 3,004 |
What methods exist for detecting multiple antigens simultaneously in a one-sample, laboratory test?
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two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology
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[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553 | 3,005 |
How many antigens could be detected by Liew's multiplex ELISA test?
|
9
|
[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553 | 3,006 |
What kind of antibodies were used in the ELISA-array assay?
|
monoclonal
|
[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553 | 3,007 |
How was the ELISA assay validated?
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using cultured viruses and inoculated chicken eggs with patient sera
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[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553 | 3,008 |
What capture antibodies were used in the study?
|
4D5, 2B5, 1F1, 2B8, 4F9, and 4E11
|
[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553 | 3,009 |
What was the spotting concentration range for the capture antibodies?
|
from 0.2 to 0.0125 mg/ml
|
[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553 | 3,010 |
How was the proper spotting concentration determined?
|
combination of minimized cross reaction and higher signal intensity
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[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553 | 3,011 |
How was cross reaction detection determined?
|
by applying JEV, YF, and DV cultures
|
[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553 | 3,012 |
How was the ELISA-array assay validated?
|
using cultured viruses and inoculated chicken eggs with patient sera
|
[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553 | 3,013 |
What method is useful in administering small molecules for systemic delivery to the body?
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Intranasal
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[
"RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.",
"Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.",
"Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.",
"Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.",
"It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .",
"Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .",
"Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.",
"Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.",
"Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.",
"Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.",
"Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.",
"In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.",
"A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.",
"For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.",
"Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.",
"Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.",
"The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.",
"In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.",
"For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.",
"The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .",
"The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.",
"Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.",
"The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .",
"Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.",
". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .",
"To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.",
"Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.",
"Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .",
"An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .",
"Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.",
"This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .",
"Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.",
"Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.",
"Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.",
"Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.",
"The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.",
"Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.",
"Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .",
"Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .",
"ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.",
"Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.",
"RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.",
"Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .",
"PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .",
"These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .",
"COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.",
"Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.",
"nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.",
"Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.",
"The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.",
"However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .",
"The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .",
"It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.",
"In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .",
"MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.",
"This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .",
"There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.",
"Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.",
"Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.",
"A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .",
"Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.",
"Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.",
"In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .",
"Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .",
"Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.",
"Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .",
"The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.",
"MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.",
"Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .",
"Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.",
"It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.",
"They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.",
"This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.",
"Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.",
"Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.",
"A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.",
"Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients."
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Why is the nasal mucosa useful in the delivery of small molecules into the body?
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the surface area can result in rapid absorption of the medication into the blood
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"RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.",
"Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.",
"Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.",
"Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.",
"It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .",
"Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .",
"Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.",
"Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.",
"Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.",
"Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.",
"Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.",
"In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.",
"A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.",
"For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.",
"Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.",
"Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.",
"The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.",
"In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.",
"For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.",
"The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .",
"The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.",
"Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.",
"The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .",
"Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.",
". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .",
"To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.",
"Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.",
"Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .",
"An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .",
"Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.",
"This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .",
"Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.",
"Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.",
"Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.",
"Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.",
"The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.",
"Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.",
"Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .",
"Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .",
"ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.",
"Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.",
"RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.",
"Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .",
"PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .",
"These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .",
"COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.",
"Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.",
"nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.",
"Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.",
"The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.",
"However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .",
"The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .",
"It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.",
"In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .",
"MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.",
"This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .",
"There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.",
"Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.",
"Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.",
"A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .",
"Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.",
"Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.",
"In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .",
"Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .",
"Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.",
"Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .",
"The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.",
"MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.",
"Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .",
"Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.",
"It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.",
"They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.",
"This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.",
"Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.",
"Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.",
"A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.",
"Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients."
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What are the most common methods of inhaled delivery of medications?
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Metered dose inhalers (MDIs) and dry powder inhalers (DPIs)
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[
"RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.",
"Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.",
"Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.",
"Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.",
"It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .",
"Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .",
"Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.",
"Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.",
"Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.",
"Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.",
"Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.",
"In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.",
"A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.",
"For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.",
"Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.",
"Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.",
"The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.",
"In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.",
"For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.",
"The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .",
"The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.",
"Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.",
"The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .",
"Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.",
". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .",
"To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.",
"Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.",
"Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .",
"An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .",
"Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.",
"This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .",
"Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.",
"Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.",
"Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.",
"Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.",
"The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.",
"Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.",
"Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .",
"Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .",
"ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.",
"Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.",
"RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.",
"Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .",
"PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .",
"These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .",
"COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.",
"Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.",
"nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.",
"Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.",
"The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.",
"However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .",
"The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .",
"It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.",
"In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .",
"MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.",
"This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .",
"There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.",
"Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.",
"Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.",
"A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .",
"Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.",
"Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.",
"In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .",
"Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .",
"Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.",
"Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .",
"The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.",
"MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.",
"Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .",
"Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.",
"It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.",
"They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.",
"This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.",
"Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.",
"Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.",
"A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.",
"Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients."
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What medications have shown good promise to in vivo delivery via dry powder inhalers?
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insulin and low-molecular-weight heparin
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"RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.",
"Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.",
"Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.",
"Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.",
"It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .",
"Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .",
"Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.",
"Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.",
"Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.",
"Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.",
"Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.",
"In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.",
"A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.",
"For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.",
"Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.",
"Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.",
"The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.",
"In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.",
"For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.",
"The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .",
"The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.",
"Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.",
"The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .",
"Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.",
". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .",
"To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.",
"Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.",
"Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .",
"An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .",
"Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.",
"This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .",
"Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.",
"Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.",
"Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.",
"Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.",
"The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.",
"Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.",
"Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .",
"Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .",
"ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.",
"Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.",
"RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.",
"Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .",
"PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .",
"These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .",
"COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.",
"Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.",
"nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.",
"Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.",
"The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.",
"However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .",
"The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .",
"It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.",
"In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .",
"MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.",
"This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .",
"There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.",
"Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.",
"Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.",
"A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .",
"Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.",
"Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.",
"In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .",
"Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .",
"Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.",
"Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .",
"The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.",
"MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.",
"Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .",
"Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.",
"It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.",
"They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.",
"This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.",
"Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.",
"Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.",
"A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.",
"Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients."
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How are siRNAs typically delivered for systemic effect?
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intratracheal or intranasal delivery
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[
"RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.",
"Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.",
"Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.",
"Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.",
"It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .",
"Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .",
"Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.",
"Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.",
"Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.",
"Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.",
"Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.",
"In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.",
"A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.",
"For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.",
"Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.",
"Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.",
"The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.",
"In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.",
"For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.",
"The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .",
"The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.",
"Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.",
"The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .",
"Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.",
". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .",
"To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.",
"Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.",
"Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .",
"An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .",
"Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.",
"This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .",
"Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.",
"Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.",
"Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.",
"Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.",
"The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.",
"Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.",
"Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .",
"Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .",
"ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.",
"Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.",
"RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.",
"Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .",
"PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .",
"These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .",
"COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.",
"Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.",
"nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.",
"Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.",
"The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.",
"However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .",
"The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .",
"It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.",
"In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .",
"MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.",
"This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .",
"There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.",
"Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.",
"Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.",
"A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .",
"Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.",
"Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.",
"In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .",
"Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .",
"Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.",
"Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .",
"The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.",
"MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.",
"Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .",
"Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.",
"It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.",
"They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.",
"This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.",
"Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.",
"Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.",
"A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.",
"Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients."
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What structures form the human airway?
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respiratory bronchioles, alveolar ducts, and alveolar sacs
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"RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.",
"Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.",
"Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.",
"Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.",
"It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .",
"Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .",
"Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.",
"Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.",
"Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.",
"Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.",
"Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.",
"In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.",
"A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.",
"For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.",
"Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.",
"Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.",
"The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.",
"In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.",
"For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.",
"The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .",
"The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.",
"Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.",
"The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .",
"Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.",
". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .",
"To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.",
"Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.",
"Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .",
"An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .",
"Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.",
"This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .",
"Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.",
"Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.",
"Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.",
"Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.",
"The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.",
"Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.",
"Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .",
"Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .",
"ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.",
"Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.",
"RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.",
"Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .",
"PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .",
"These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .",
"COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.",
"Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.",
"nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.",
"Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.",
"The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.",
"However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .",
"The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .",
"It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.",
"In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .",
"MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.",
"This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .",
"There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.",
"Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.",
"Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.",
"A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .",
"Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.",
"Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.",
"In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .",
"Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .",
"Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.",
"Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .",
"The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.",
"MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.",
"Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .",
"Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.",
"It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.",
"They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.",
"This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.",
"Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.",
"Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.",
"A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.",
"Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients."
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What size of particle has been shown to be most effective in the delivery to the lower airway?
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"RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.",
"Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.",
"Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.",
"Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.",
"It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .",
"Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .",
"Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.",
"Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.",
"Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.",
"Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.",
"Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.",
"In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.",
"A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.",
"For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.",
"Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.",
"Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.",
"The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.",
"In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.",
"For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.",
"The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .",
"The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.",
"Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.",
"The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .",
"Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.",
". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .",
"To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.",
"Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.",
"Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .",
"An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .",
"Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.",
"This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .",
"Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.",
"Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.",
"Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.",
"Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.",
"The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.",
"Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.",
"Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .",
"Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .",
"ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.",
"Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.",
"RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.",
"Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .",
"PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .",
"These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .",
"COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.",
"Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.",
"nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.",
"Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.",
"The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.",
"However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .",
"The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .",
"It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.",
"In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .",
"MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.",
"This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .",
"There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.",
"Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.",
"Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.",
"A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .",
"Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.",
"Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.",
"In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .",
"Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .",
"Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.",
"Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .",
"The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.",
"MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.",
"Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .",
"Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.",
"It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.",
"They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.",
"This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.",
"Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.",
"Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.",
"A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.",
"Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients."
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What are the essential conditions in siRNA delivery to effectively produce gene silencing in the lungs?
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delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration
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"RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.",
"Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.",
"Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.",
"Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.",
"It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .",
"Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .",
"Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.",
"Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.",
"Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.",
"Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.",
"Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.",
"In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.",
"A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.",
"For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.",
"Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.",
"Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.",
"The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.",
"In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.",
"For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.",
"The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .",
"The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.",
"Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.",
"The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .",
"Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.",
". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .",
"To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.",
"Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.",
"Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .",
"An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .",
"Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.",
"This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .",
"Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.",
"Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.",
"Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.",
"Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.",
"The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.",
"Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.",
"Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .",
"Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .",
"ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.",
"Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.",
"RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.",
"Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .",
"PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .",
"These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .",
"COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.",
"Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.",
"nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.",
"Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.",
"The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.",
"However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .",
"The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .",
"It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.",
"In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .",
"MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.",
"This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .",
"There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.",
"Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.",
"Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.",
"A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .",
"Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.",
"Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.",
"In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .",
"Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .",
"Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.",
"Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .",
"The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.",
"MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.",
"Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .",
"Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.",
"It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.",
"They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.",
"This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.",
"Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.",
"Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.",
"A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.",
"Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients."
] | 641 | 1,621 |
How long is the SAIBK gene?
|
27,534 nucleotides
|
[
"Infectious bronchitis virus IBV causes tremendous economic losses to the poultry industry. Here, we report the complete genome analysis results for a new natural recombination nephropathogenic IBV strain named SAIBK, which was isolated in the Sichuan province of China in 2005. Text: tagious and acute disease in domestic chickens, belongs to group III of the genus Coronavirus in the family Coronaviridae . . It is an enveloped, unsegmented, positive-sense, single-stranded RNA ssRNA virus and has a genome of approximately 27.6 kb . . Recently, many epidemiological analysis reports have suggested that nephropathogenic IBVs have become increasingly prevalent . . . . in China.",
". Recently, many epidemiological analysis reports have suggested that nephropathogenic IBVs have become increasingly prevalent . . . . in China. In this work, the complete genome sequence of an isolate named SAIBK was analyzed and recombination was detected between SAIBK and some previously reported IBVs. A rapid amplification of cDNA ends RACE kit TaKaRa, Japan was used to obtain the 5= and 3= ends of the genome. Other parts were amplified by 19 primers with overlap between each fragment and were cloned into the pMD19-T vector TaKaRa, Japan . All fragments were sequenced three times by Sangon Biotech Shanghai, China . The sequenced fragments were assembled using the SeqMan software program DNAStar, Inc. .",
"All fragments were sequenced three times by Sangon Biotech Shanghai, China . The sequenced fragments were assembled using the SeqMan software program DNAStar, Inc. . Sequence alignment was conducted and a phylogenetic tree was constructed using the software program MEGA5 .. Recombination analysis was performed using the RDP 4.14 . and SimPlot 3.5.1 . software programs. The complete genome of the SAIBK strain is 27,534 nucleotides nt in length, including the poly A tail. It has a classical IBV genome organization with 10 open reading frames ORFs : The genome sequence of SAIBK shows the highest identity 94.3% to the Chinese IBV strain SC021202 GenBank accession no.",
"The complete genome of the SAIBK strain is 27,534 nucleotides nt in length, including the poly A tail. It has a classical IBV genome organization with 10 open reading frames ORFs : The genome sequence of SAIBK shows the highest identity 94.3% to the Chinese IBV strain SC021202 GenBank accession no. EU714029 and the lowest identity 85.8% to two Chinese IBV strains, BJ GenBank accession no. AY319651 and DY07 GenBank accession no. HM245923 . It has lower nucleotide identities of 88.1%, 87.9%, and 87.7% to the most popularly used IBV vaccine strains, H120, H52, and M41, respectively.",
"HM245923 . It has lower nucleotide identities of 88.1%, 87.9%, and 87.7% to the most popularly used IBV vaccine strains, H120, H52, and M41, respectively. Phylogenetic analysis of the complete genome results indicated that the SAIBK strain clusters into the same branch as the IBV YN strain GenBank accession no. JF893452 and the SC021202 strain GenBank accession no. EU714029 . The S1 subunit of the IBV genome is the major determinant of serotype . . . . , and S1 analysis indicated that the SAIBK strain has a 4/91-like serotype.",
"The S1 subunit of the IBV genome is the major determinant of serotype . . . . , and S1 analysis indicated that the SAIBK strain has a 4/91-like serotype. The employed recombination detection methods revealed that SAIBK is a chimera virus, with recombination by the SC021202 strain as a major parent and the H120 vaccine strain as a minor parent. The first and second recombination regions were located at positions 7231 to 9126 and 13437 to 14473 in genes 1a and 1b, respectively.",
"The employed recombination detection methods revealed that SAIBK is a chimera virus, with recombination by the SC021202 strain as a major parent and the H120 vaccine strain as a minor parent. The first and second recombination regions were located at positions 7231 to 9126 and 13437 to 14473 in genes 1a and 1b, respectively. There were two other recombination regions detected at positions 951 to 1067 and 5393 to 5605 of SAIBK, which were recombined with the SC021202 strain as a major parent and the H52 vaccine strain as a minor parent. The recombination detection results suggested that SAIBK is possibly a chimera virus derived from the popularly used vaccine strains H120 and H52 and the field strain SC021202, and the SC021202 strain was isolated from chickens vaccinated with H120 in the Sichuan province of China in 2003 . . This result revealed that the field IBVs in Sichuan Province have undergone genetic recombination and are possibly emerging as new mutant strains, such as SAIBK.",
". This result revealed that the field IBVs in Sichuan Province have undergone genetic recombination and are possibly emerging as new mutant strains, such as SAIBK. Nucleotide sequence accession number. The complete genome sequence of the SAIBK isolate was submitted to GenBank and assigned the accession no. DQ288927."
] | 1,547 | 1,622 |
How many open reading frames are in the SAIBK gene?
|
10
|
[
"Infectious bronchitis virus IBV causes tremendous economic losses to the poultry industry. Here, we report the complete genome analysis results for a new natural recombination nephropathogenic IBV strain named SAIBK, which was isolated in the Sichuan province of China in 2005. Text: tagious and acute disease in domestic chickens, belongs to group III of the genus Coronavirus in the family Coronaviridae . . It is an enveloped, unsegmented, positive-sense, single-stranded RNA ssRNA virus and has a genome of approximately 27.6 kb . . Recently, many epidemiological analysis reports have suggested that nephropathogenic IBVs have become increasingly prevalent . . . . in China.",
". Recently, many epidemiological analysis reports have suggested that nephropathogenic IBVs have become increasingly prevalent . . . . in China. In this work, the complete genome sequence of an isolate named SAIBK was analyzed and recombination was detected between SAIBK and some previously reported IBVs. A rapid amplification of cDNA ends RACE kit TaKaRa, Japan was used to obtain the 5= and 3= ends of the genome. Other parts were amplified by 19 primers with overlap between each fragment and were cloned into the pMD19-T vector TaKaRa, Japan . All fragments were sequenced three times by Sangon Biotech Shanghai, China . The sequenced fragments were assembled using the SeqMan software program DNAStar, Inc. .",
"All fragments were sequenced three times by Sangon Biotech Shanghai, China . The sequenced fragments were assembled using the SeqMan software program DNAStar, Inc. . Sequence alignment was conducted and a phylogenetic tree was constructed using the software program MEGA5 .. Recombination analysis was performed using the RDP 4.14 . and SimPlot 3.5.1 . software programs. The complete genome of the SAIBK strain is 27,534 nucleotides nt in length, including the poly A tail. It has a classical IBV genome organization with 10 open reading frames ORFs : The genome sequence of SAIBK shows the highest identity 94.3% to the Chinese IBV strain SC021202 GenBank accession no.",
"The complete genome of the SAIBK strain is 27,534 nucleotides nt in length, including the poly A tail. It has a classical IBV genome organization with 10 open reading frames ORFs : The genome sequence of SAIBK shows the highest identity 94.3% to the Chinese IBV strain SC021202 GenBank accession no. EU714029 and the lowest identity 85.8% to two Chinese IBV strains, BJ GenBank accession no. AY319651 and DY07 GenBank accession no. HM245923 . It has lower nucleotide identities of 88.1%, 87.9%, and 87.7% to the most popularly used IBV vaccine strains, H120, H52, and M41, respectively.",
"HM245923 . It has lower nucleotide identities of 88.1%, 87.9%, and 87.7% to the most popularly used IBV vaccine strains, H120, H52, and M41, respectively. Phylogenetic analysis of the complete genome results indicated that the SAIBK strain clusters into the same branch as the IBV YN strain GenBank accession no. JF893452 and the SC021202 strain GenBank accession no. EU714029 . The S1 subunit of the IBV genome is the major determinant of serotype . . . . , and S1 analysis indicated that the SAIBK strain has a 4/91-like serotype.",
"The S1 subunit of the IBV genome is the major determinant of serotype . . . . , and S1 analysis indicated that the SAIBK strain has a 4/91-like serotype. The employed recombination detection methods revealed that SAIBK is a chimera virus, with recombination by the SC021202 strain as a major parent and the H120 vaccine strain as a minor parent. The first and second recombination regions were located at positions 7231 to 9126 and 13437 to 14473 in genes 1a and 1b, respectively.",
"The employed recombination detection methods revealed that SAIBK is a chimera virus, with recombination by the SC021202 strain as a major parent and the H120 vaccine strain as a minor parent. The first and second recombination regions were located at positions 7231 to 9126 and 13437 to 14473 in genes 1a and 1b, respectively. There were two other recombination regions detected at positions 951 to 1067 and 5393 to 5605 of SAIBK, which were recombined with the SC021202 strain as a major parent and the H52 vaccine strain as a minor parent. The recombination detection results suggested that SAIBK is possibly a chimera virus derived from the popularly used vaccine strains H120 and H52 and the field strain SC021202, and the SC021202 strain was isolated from chickens vaccinated with H120 in the Sichuan province of China in 2003 . . This result revealed that the field IBVs in Sichuan Province have undergone genetic recombination and are possibly emerging as new mutant strains, such as SAIBK.",
". This result revealed that the field IBVs in Sichuan Province have undergone genetic recombination and are possibly emerging as new mutant strains, such as SAIBK. Nucleotide sequence accession number. The complete genome sequence of the SAIBK isolate was submitted to GenBank and assigned the accession no. DQ288927."
] | 1,547 | 1,623 |
What virus has the closest genetic identity with the SAIBK gene?
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Chinese IBV strain SC021202
|
[
"Infectious bronchitis virus IBV causes tremendous economic losses to the poultry industry. Here, we report the complete genome analysis results for a new natural recombination nephropathogenic IBV strain named SAIBK, which was isolated in the Sichuan province of China in 2005. Text: tagious and acute disease in domestic chickens, belongs to group III of the genus Coronavirus in the family Coronaviridae . . It is an enveloped, unsegmented, positive-sense, single-stranded RNA ssRNA virus and has a genome of approximately 27.6 kb . . Recently, many epidemiological analysis reports have suggested that nephropathogenic IBVs have become increasingly prevalent . . . . in China.",
". Recently, many epidemiological analysis reports have suggested that nephropathogenic IBVs have become increasingly prevalent . . . . in China. In this work, the complete genome sequence of an isolate named SAIBK was analyzed and recombination was detected between SAIBK and some previously reported IBVs. A rapid amplification of cDNA ends RACE kit TaKaRa, Japan was used to obtain the 5= and 3= ends of the genome. Other parts were amplified by 19 primers with overlap between each fragment and were cloned into the pMD19-T vector TaKaRa, Japan . All fragments were sequenced three times by Sangon Biotech Shanghai, China . The sequenced fragments were assembled using the SeqMan software program DNAStar, Inc. .",
"All fragments were sequenced three times by Sangon Biotech Shanghai, China . The sequenced fragments were assembled using the SeqMan software program DNAStar, Inc. . Sequence alignment was conducted and a phylogenetic tree was constructed using the software program MEGA5 .. Recombination analysis was performed using the RDP 4.14 . and SimPlot 3.5.1 . software programs. The complete genome of the SAIBK strain is 27,534 nucleotides nt in length, including the poly A tail. It has a classical IBV genome organization with 10 open reading frames ORFs : The genome sequence of SAIBK shows the highest identity 94.3% to the Chinese IBV strain SC021202 GenBank accession no.",
"The complete genome of the SAIBK strain is 27,534 nucleotides nt in length, including the poly A tail. It has a classical IBV genome organization with 10 open reading frames ORFs : The genome sequence of SAIBK shows the highest identity 94.3% to the Chinese IBV strain SC021202 GenBank accession no. EU714029 and the lowest identity 85.8% to two Chinese IBV strains, BJ GenBank accession no. AY319651 and DY07 GenBank accession no. HM245923 . It has lower nucleotide identities of 88.1%, 87.9%, and 87.7% to the most popularly used IBV vaccine strains, H120, H52, and M41, respectively.",
"HM245923 . It has lower nucleotide identities of 88.1%, 87.9%, and 87.7% to the most popularly used IBV vaccine strains, H120, H52, and M41, respectively. Phylogenetic analysis of the complete genome results indicated that the SAIBK strain clusters into the same branch as the IBV YN strain GenBank accession no. JF893452 and the SC021202 strain GenBank accession no. EU714029 . The S1 subunit of the IBV genome is the major determinant of serotype . . . . , and S1 analysis indicated that the SAIBK strain has a 4/91-like serotype.",
"The S1 subunit of the IBV genome is the major determinant of serotype . . . . , and S1 analysis indicated that the SAIBK strain has a 4/91-like serotype. The employed recombination detection methods revealed that SAIBK is a chimera virus, with recombination by the SC021202 strain as a major parent and the H120 vaccine strain as a minor parent. The first and second recombination regions were located at positions 7231 to 9126 and 13437 to 14473 in genes 1a and 1b, respectively.",
"The employed recombination detection methods revealed that SAIBK is a chimera virus, with recombination by the SC021202 strain as a major parent and the H120 vaccine strain as a minor parent. The first and second recombination regions were located at positions 7231 to 9126 and 13437 to 14473 in genes 1a and 1b, respectively. There were two other recombination regions detected at positions 951 to 1067 and 5393 to 5605 of SAIBK, which were recombined with the SC021202 strain as a major parent and the H52 vaccine strain as a minor parent. The recombination detection results suggested that SAIBK is possibly a chimera virus derived from the popularly used vaccine strains H120 and H52 and the field strain SC021202, and the SC021202 strain was isolated from chickens vaccinated with H120 in the Sichuan province of China in 2003 . . This result revealed that the field IBVs in Sichuan Province have undergone genetic recombination and are possibly emerging as new mutant strains, such as SAIBK.",
". This result revealed that the field IBVs in Sichuan Province have undergone genetic recombination and are possibly emerging as new mutant strains, such as SAIBK. Nucleotide sequence accession number. The complete genome sequence of the SAIBK isolate was submitted to GenBank and assigned the accession no. DQ288927."
] | 1,547 | 1,624 |
What is the leading cause of death among children after the age of 1 month?
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Pneumonia remains the leading cause of death in children outside the neonatal period,
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[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
] | 1,571 | 502 |
How has the number of childhood pneumonia been reduced?
|
New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases
|
[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
] | 1,571 | 504 |
What percentage of childhood deaths are due to pneumonia?
|
approximately 900,000 of the estimated 6.3 million child deaths in 2013
|
[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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How has the childhood population grown in the last two decades?
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global childhood population from 605 million in 2000 to 664 million in 2015
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What is the reduction in the number of childhood pneumonia cases?
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Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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How much is the reduction in the childhood pneumonia deaths?
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Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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Childhood pneumonia rate for high income countries vs low and middle income countries.
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The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries.
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What percentage of childhood pneumonia deaths occur outside hospital in low and middle income countries?
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up to 81% of severe pneumonia deaths occur outside a hospital
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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Case Fatality Rates for Childhood Pneumonia in high income vs low and middle income countries.
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the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries
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[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
] | 1,571 | 515 |
How can childhood pneumonia affect the subsequent health of a person?
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Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
] | 1,571 | 516 |
What is the increase in the risk of respiratory disease after having childhood pneumonia.
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The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.
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[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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Which is the best method to identify pneumonia in a person?
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Chest radiologic changes have been considered the gold standard for defining a pneumonia event
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What is responsible for the reduction of radiologic pneumonia?
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Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia.
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What is the percentage reduction in pneumonia cases due to vaccination?
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Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What is the revised WHO definition of Bacterial Pneumonia?
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A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What is the reduction in bacterial pneumonia under the revised WHO definition of bacterial pneumonia?
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Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What caused the increase in the incidence of empyema in children in the recent past?
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An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
] | 1,571 | 530 |
How have the incidence Empyema been reduced?
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These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years
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[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
] | 1,571 | 532 |
What pneumonia related or chest conditions indicate the need for child radiography?
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chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications.
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[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What chest diseases and pneumonia were identified as leading causes prior to the availability of vaccines?
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prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus
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[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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Why has pertussis immunity in infants has decreased in infants?
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Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What is the effect of childhood tuberculosis in childhood pneumonia?
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A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What are the risk factors in childhood pneumonia?
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incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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How does air pollution affect the incidence of childhood pneumonia?
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Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What is the strongest risk factor for childhood pneumonia?
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The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What is the global coverage of influenza and pneumonia vaccines?
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By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
] | 1,571 | 540 |
Is influenza vaccination during pregnancy safe? How long does it protect the child?
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Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh and 50.4% in South Africa ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks
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[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
] | 1,571 | 541 |
What is emphyema?
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a rare complication of pneumonia
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[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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How is the term end point consolidation described with regard to pneumonia diagnosis?
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as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What factors make H5N1 a worldwide threat to public health?
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rapid spread and high pathogenicity
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"The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide EAP against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection.",
"We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS .",
"Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths a mortality rate >50% , from 2003 to 2013 influenza/human animal interface/H5N1 cumulative table archives/en/index.html . Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation .",
"Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests .",
"This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP .",
"In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP . The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza.",
"In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010.",
"Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney MDCK cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water.",
"8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney MDCK cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China.",
"The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator.",
"The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol v/v and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method .",
"The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method . Vitro. 2.5 mL A549 and Raw 264.7 cells 4 × 10 5 /mL per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP 50, 100, 200 g/mL was added to each well. Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR .",
"Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR . Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA.",
"Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay.",
"Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM Hyclone Laboratories, Logan, UT, USA containing 10% FBS Hyclone Laboratories , 100 U/mL penicillin, and 100 g/mL streptomycin Invitrogen, San Diego, CA, USA . Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet.",
"Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol Invitrogen according to the manufacturer's instructions. DNaseItreated RNA 0.2 g was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 .",
"The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 Applied Biosystems using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard .",
"The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm.",
"IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 SPSS Taiwan Corp., Taiwan , and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells.",
"Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP 50, 100, 200 g/mL for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU.",
"Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 a , all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant.",
"Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice Figure 2 b . To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d .",
"To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d . These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c .",
"To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c . Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group = 0.0599 Figures 3 g -3 i . These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f .",
"IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f . These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d .",
"To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d . The lungs of EAP 25 mg/kg treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels Figures 4 a and 4 b . Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs .",
"Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs . To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 TLR2 , TLR4, Dectin-1, and mannose receptor MR were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a .",
"Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a . In vitro assay showed similar trends. As shown in Figure 5 b , Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model.",
"These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro.",
"The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza .",
"Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus including H1N1 and H3N2 subtypes . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro.",
"The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment 25 mg/kg body weight was significantly higher than in mice receiving PBS 50% to 0% . In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury.",
"In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect . However, it is unclear whether the lack of proinflammatory cytokines such as TNFand IL-6 facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections .",
"Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive .",
"Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage data not show . The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival .",
"The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival . TNF-has been reported to exert a defensive effect against influenza infection in vitro . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS.",
"In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response.",
"In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro.",
"In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection.",
"Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products."
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What are the symptoms of H5N1 infection in humans?
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fever, encephalitis, pneumonia, and severe acute respiratory syndrome (SARS)
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[
"The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide EAP against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection.",
"We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS .",
"Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths a mortality rate >50% , from 2003 to 2013 influenza/human animal interface/H5N1 cumulative table archives/en/index.html . Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation .",
"Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests .",
"This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP .",
"In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP . The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza.",
"In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010.",
"Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney MDCK cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water.",
"8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney MDCK cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China.",
"The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator.",
"The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol v/v and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method .",
"The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method . Vitro. 2.5 mL A549 and Raw 264.7 cells 4 × 10 5 /mL per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP 50, 100, 200 g/mL was added to each well. Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR .",
"Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR . Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA.",
"Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay.",
"Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM Hyclone Laboratories, Logan, UT, USA containing 10% FBS Hyclone Laboratories , 100 U/mL penicillin, and 100 g/mL streptomycin Invitrogen, San Diego, CA, USA . Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet.",
"Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol Invitrogen according to the manufacturer's instructions. DNaseItreated RNA 0.2 g was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 .",
"The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 Applied Biosystems using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard .",
"The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm.",
"IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 SPSS Taiwan Corp., Taiwan , and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells.",
"Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP 50, 100, 200 g/mL for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU.",
"Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 a , all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant.",
"Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice Figure 2 b . To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d .",
"To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d . These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c .",
"To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c . Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group = 0.0599 Figures 3 g -3 i . These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f .",
"IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f . These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d .",
"To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d . The lungs of EAP 25 mg/kg treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels Figures 4 a and 4 b . Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs .",
"Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs . To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 TLR2 , TLR4, Dectin-1, and mannose receptor MR were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a .",
"Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a . In vitro assay showed similar trends. As shown in Figure 5 b , Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model.",
"These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro.",
"The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza .",
"Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus including H1N1 and H3N2 subtypes . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro.",
"The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment 25 mg/kg body weight was significantly higher than in mice receiving PBS 50% to 0% . In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury.",
"In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect . However, it is unclear whether the lack of proinflammatory cytokines such as TNFand IL-6 facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections .",
"Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive .",
"Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage data not show . The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival .",
"The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival . TNF-has been reported to exert a defensive effect against influenza infection in vitro . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS.",
"In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response.",
"In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro.",
"In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection.",
"Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products."
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Name some medications used to treat influenza.
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zanamivir (Relenza) and oseltamivir (Tamiflu)
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[
"The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide EAP against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection.",
"We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS .",
"Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths a mortality rate >50% , from 2003 to 2013 influenza/human animal interface/H5N1 cumulative table archives/en/index.html . Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation .",
"Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests .",
"This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP .",
"In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP . The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza.",
"In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010.",
"Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney MDCK cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water.",
"8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney MDCK cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China.",
"The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator.",
"The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol v/v and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method .",
"The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method . Vitro. 2.5 mL A549 and Raw 264.7 cells 4 × 10 5 /mL per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP 50, 100, 200 g/mL was added to each well. Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR .",
"Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR . Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA.",
"Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay.",
"Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM Hyclone Laboratories, Logan, UT, USA containing 10% FBS Hyclone Laboratories , 100 U/mL penicillin, and 100 g/mL streptomycin Invitrogen, San Diego, CA, USA . Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet.",
"Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol Invitrogen according to the manufacturer's instructions. DNaseItreated RNA 0.2 g was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 .",
"The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 Applied Biosystems using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard .",
"The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm.",
"IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 SPSS Taiwan Corp., Taiwan , and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells.",
"Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP 50, 100, 200 g/mL for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU.",
"Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 a , all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant.",
"Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice Figure 2 b . To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d .",
"To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d . These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c .",
"To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c . Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group = 0.0599 Figures 3 g -3 i . These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f .",
"IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f . These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d .",
"To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d . The lungs of EAP 25 mg/kg treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels Figures 4 a and 4 b . Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs .",
"Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs . To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 TLR2 , TLR4, Dectin-1, and mannose receptor MR were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a .",
"Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a . In vitro assay showed similar trends. As shown in Figure 5 b , Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model.",
"These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro.",
"The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza .",
"Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus including H1N1 and H3N2 subtypes . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro.",
"The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment 25 mg/kg body weight was significantly higher than in mice receiving PBS 50% to 0% . In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury.",
"In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect . However, it is unclear whether the lack of proinflammatory cytokines such as TNFand IL-6 facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections .",
"Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive .",
"Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage data not show . The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival .",
"The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival . TNF-has been reported to exert a defensive effect against influenza infection in vitro . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS.",
"In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response.",
"In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro.",
"In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection.",
"Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products."
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Why have antiretrovirals medications had limited benefit in treating influenza?
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drug-resistance and frequent antigenic mutation
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[
"The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide EAP against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection.",
"We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS .",
"Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths a mortality rate >50% , from 2003 to 2013 influenza/human animal interface/H5N1 cumulative table archives/en/index.html . Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation .",
"Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests .",
"This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP .",
"In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP . The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza.",
"In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010.",
"Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney MDCK cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water.",
"8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney MDCK cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China.",
"The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator.",
"The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol v/v and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method .",
"The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method . Vitro. 2.5 mL A549 and Raw 264.7 cells 4 × 10 5 /mL per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP 50, 100, 200 g/mL was added to each well. Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR .",
"Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR . Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA.",
"Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay.",
"Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM Hyclone Laboratories, Logan, UT, USA containing 10% FBS Hyclone Laboratories , 100 U/mL penicillin, and 100 g/mL streptomycin Invitrogen, San Diego, CA, USA . Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet.",
"Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol Invitrogen according to the manufacturer's instructions. DNaseItreated RNA 0.2 g was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 .",
"The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 Applied Biosystems using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard .",
"The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm.",
"IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 SPSS Taiwan Corp., Taiwan , and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells.",
"Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP 50, 100, 200 g/mL for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU.",
"Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 a , all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant.",
"Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice Figure 2 b . To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d .",
"To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d . These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c .",
"To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c . Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group = 0.0599 Figures 3 g -3 i . These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f .",
"IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f . These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d .",
"To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d . The lungs of EAP 25 mg/kg treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels Figures 4 a and 4 b . Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs .",
"Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs . To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 TLR2 , TLR4, Dectin-1, and mannose receptor MR were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a .",
"Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a . In vitro assay showed similar trends. As shown in Figure 5 b , Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model.",
"These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro.",
"The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza .",
"Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus including H1N1 and H3N2 subtypes . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro.",
"The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment 25 mg/kg body weight was significantly higher than in mice receiving PBS 50% to 0% . In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury.",
"In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect . However, it is unclear whether the lack of proinflammatory cytokines such as TNFand IL-6 facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections .",
"Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive .",
"Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage data not show . The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival .",
"The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival . TNF-has been reported to exert a defensive effect against influenza infection in vitro . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS.",
"In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response.",
"In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro.",
"In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection.",
"Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products."
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What is the focus of the current study?
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effect of EAP against H5N1 influenza infection
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"The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide EAP against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection.",
"We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS .",
"Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths a mortality rate >50% , from 2003 to 2013 influenza/human animal interface/H5N1 cumulative table archives/en/index.html . Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation .",
"Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests .",
"This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP .",
"In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP . The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza.",
"In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010.",
"Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney MDCK cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water.",
"8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney MDCK cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China.",
"The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator.",
"The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol v/v and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method .",
"The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method . Vitro. 2.5 mL A549 and Raw 264.7 cells 4 × 10 5 /mL per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP 50, 100, 200 g/mL was added to each well. Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR .",
"Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR . Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA.",
"Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay.",
"Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM Hyclone Laboratories, Logan, UT, USA containing 10% FBS Hyclone Laboratories , 100 U/mL penicillin, and 100 g/mL streptomycin Invitrogen, San Diego, CA, USA . Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet.",
"Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol Invitrogen according to the manufacturer's instructions. DNaseItreated RNA 0.2 g was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 .",
"The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 Applied Biosystems using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard .",
"The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm.",
"IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 SPSS Taiwan Corp., Taiwan , and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells.",
"Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP 50, 100, 200 g/mL for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU.",
"Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 a , all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant.",
"Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice Figure 2 b . To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d .",
"To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d . These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c .",
"To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c . Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group = 0.0599 Figures 3 g -3 i . These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f .",
"IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f . These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d .",
"To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d . The lungs of EAP 25 mg/kg treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels Figures 4 a and 4 b . Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs .",
"Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs . To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 TLR2 , TLR4, Dectin-1, and mannose receptor MR were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a .",
"Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a . In vitro assay showed similar trends. As shown in Figure 5 b , Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model.",
"These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro.",
"The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza .",
"Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus including H1N1 and H3N2 subtypes . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro.",
"The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment 25 mg/kg body weight was significantly higher than in mice receiving PBS 50% to 0% . In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury.",
"In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect . However, it is unclear whether the lack of proinflammatory cytokines such as TNFand IL-6 facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections .",
"Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive .",
"Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage data not show . The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival .",
"The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival . TNF-has been reported to exert a defensive effect against influenza infection in vitro . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS.",
"In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response.",
"In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro.",
"In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection.",
"Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products."
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What is the result of the current study?
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the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents
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"The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide EAP against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection.",
"We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS .",
"Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths a mortality rate >50% , from 2003 to 2013 influenza/human animal interface/H5N1 cumulative table archives/en/index.html . Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation .",
"Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests .",
"This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP .",
"In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP . The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza.",
"In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010.",
"Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney MDCK cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water.",
"8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney MDCK cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China.",
"The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator.",
"The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol v/v and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method .",
"The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method . Vitro. 2.5 mL A549 and Raw 264.7 cells 4 × 10 5 /mL per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP 50, 100, 200 g/mL was added to each well. Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR .",
"Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR . Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA.",
"Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay.",
"Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM Hyclone Laboratories, Logan, UT, USA containing 10% FBS Hyclone Laboratories , 100 U/mL penicillin, and 100 g/mL streptomycin Invitrogen, San Diego, CA, USA . Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet.",
"Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol Invitrogen according to the manufacturer's instructions. DNaseItreated RNA 0.2 g was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 .",
"The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 Applied Biosystems using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard .",
"The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm.",
"IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 SPSS Taiwan Corp., Taiwan , and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells.",
"Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP 50, 100, 200 g/mL for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU.",
"Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 a , all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant.",
"Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice Figure 2 b . To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d .",
"To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d . These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c .",
"To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c . Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group = 0.0599 Figures 3 g -3 i . These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f .",
"IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f . These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d .",
"To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d . The lungs of EAP 25 mg/kg treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels Figures 4 a and 4 b . Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs .",
"Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs . To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 TLR2 , TLR4, Dectin-1, and mannose receptor MR were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a .",
"Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a . In vitro assay showed similar trends. As shown in Figure 5 b , Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model.",
"These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro.",
"The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza .",
"Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus including H1N1 and H3N2 subtypes . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro.",
"The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment 25 mg/kg body weight was significantly higher than in mice receiving PBS 50% to 0% . In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury.",
"In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect . However, it is unclear whether the lack of proinflammatory cytokines such as TNFand IL-6 facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections .",
"Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive .",
"Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage data not show . The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival .",
"The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival . TNF-has been reported to exert a defensive effect against influenza infection in vitro . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS.",
"In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response.",
"In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro.",
"In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection.",
"Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products."
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How do the polysaccharides in plants effect the immune response?
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enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12
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[
"The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide EAP against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection.",
"We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS .",
"Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths a mortality rate >50% , from 2003 to 2013 influenza/human animal interface/H5N1 cumulative table archives/en/index.html . Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation .",
"Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests .",
"This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP .",
"In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP . The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza.",
"In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010.",
"Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney MDCK cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water.",
"8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney MDCK cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China.",
"The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator.",
"The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol v/v and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method .",
"The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method . Vitro. 2.5 mL A549 and Raw 264.7 cells 4 × 10 5 /mL per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP 50, 100, 200 g/mL was added to each well. Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR .",
"Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR . Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA.",
"Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay.",
"Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM Hyclone Laboratories, Logan, UT, USA containing 10% FBS Hyclone Laboratories , 100 U/mL penicillin, and 100 g/mL streptomycin Invitrogen, San Diego, CA, USA . Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet.",
"Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol Invitrogen according to the manufacturer's instructions. DNaseItreated RNA 0.2 g was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 .",
"The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 Applied Biosystems using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard .",
"The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm.",
"IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 SPSS Taiwan Corp., Taiwan , and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells.",
"Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP 50, 100, 200 g/mL for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU.",
"Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 a , all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant.",
"Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice Figure 2 b . To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d .",
"To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d . These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c .",
"To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c . Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group = 0.0599 Figures 3 g -3 i . These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f .",
"IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f . These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d .",
"To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d . The lungs of EAP 25 mg/kg treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels Figures 4 a and 4 b . Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs .",
"Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs . To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 TLR2 , TLR4, Dectin-1, and mannose receptor MR were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a .",
"Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a . In vitro assay showed similar trends. As shown in Figure 5 b , Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model.",
"These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro.",
"The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza .",
"Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus including H1N1 and H3N2 subtypes . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro.",
"The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment 25 mg/kg body weight was significantly higher than in mice receiving PBS 50% to 0% . In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury.",
"In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect . However, it is unclear whether the lack of proinflammatory cytokines such as TNFand IL-6 facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections .",
"Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive .",
"Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage data not show . The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival .",
"The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival . TNF-has been reported to exert a defensive effect against influenza infection in vitro . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS.",
"In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response.",
"In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro.",
"In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection.",
"Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products."
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What does this study demonstrate?
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EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection
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"The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide EAP against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection.",
"We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS .",
"Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths a mortality rate >50% , from 2003 to 2013 influenza/human animal interface/H5N1 cumulative table archives/en/index.html . Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation .",
"Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests .",
"This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP .",
"In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP . The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza.",
"In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010.",
"Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney MDCK cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water.",
"8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney MDCK cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China.",
"The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator.",
"The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol v/v and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method .",
"The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method . Vitro. 2.5 mL A549 and Raw 264.7 cells 4 × 10 5 /mL per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP 50, 100, 200 g/mL was added to each well. Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR .",
"Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR . Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA.",
"Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay.",
"Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM Hyclone Laboratories, Logan, UT, USA containing 10% FBS Hyclone Laboratories , 100 U/mL penicillin, and 100 g/mL streptomycin Invitrogen, San Diego, CA, USA . Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet.",
"Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol Invitrogen according to the manufacturer's instructions. DNaseItreated RNA 0.2 g was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 .",
"The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 Applied Biosystems using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard .",
"The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm.",
"IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 SPSS Taiwan Corp., Taiwan , and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells.",
"Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP 50, 100, 200 g/mL for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU.",
"Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 a , all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant.",
"Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice Figure 2 b . To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d .",
"To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d . These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c .",
"To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c . Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group = 0.0599 Figures 3 g -3 i . These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f .",
"IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f . These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d .",
"To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d . The lungs of EAP 25 mg/kg treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels Figures 4 a and 4 b . Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs .",
"Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs . To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 TLR2 , TLR4, Dectin-1, and mannose receptor MR were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a .",
"Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a . In vitro assay showed similar trends. As shown in Figure 5 b , Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model.",
"These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro.",
"The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza .",
"Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus including H1N1 and H3N2 subtypes . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro.",
"The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment 25 mg/kg body weight was significantly higher than in mice receiving PBS 50% to 0% . In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury.",
"In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect . However, it is unclear whether the lack of proinflammatory cytokines such as TNFand IL-6 facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections .",
"Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive .",
"Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage data not show . The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival .",
"The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival . TNF-has been reported to exert a defensive effect against influenza infection in vitro . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS.",
"In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response.",
"In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro.",
"In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection.",
"Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products."
] | 1,585 | 5,224 |
In this study, how did treatment of EAP after infection affect survival?
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did not provide a survival advantage
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[
"The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide EAP against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection.",
"We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS .",
"Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths a mortality rate >50% , from 2003 to 2013 influenza/human animal interface/H5N1 cumulative table archives/en/index.html . Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation .",
"Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests .",
"This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP .",
"In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP . The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza.",
"In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010.",
"Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney MDCK cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water.",
"8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney MDCK cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China.",
"The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator.",
"The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol v/v and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method .",
"The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method . Vitro. 2.5 mL A549 and Raw 264.7 cells 4 × 10 5 /mL per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP 50, 100, 200 g/mL was added to each well. Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR .",
"Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR . Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA.",
"Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay.",
"Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM Hyclone Laboratories, Logan, UT, USA containing 10% FBS Hyclone Laboratories , 100 U/mL penicillin, and 100 g/mL streptomycin Invitrogen, San Diego, CA, USA . Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet.",
"Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol Invitrogen according to the manufacturer's instructions. DNaseItreated RNA 0.2 g was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 .",
"The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 Applied Biosystems using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard .",
"The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm.",
"IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 SPSS Taiwan Corp., Taiwan , and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells.",
"Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP 50, 100, 200 g/mL for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU.",
"Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 a , all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant.",
"Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice Figure 2 b . To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d .",
"To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d . These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c .",
"To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c . Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group = 0.0599 Figures 3 g -3 i . These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f .",
"IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f . These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d .",
"To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d . The lungs of EAP 25 mg/kg treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels Figures 4 a and 4 b . Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs .",
"Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs . To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 TLR2 , TLR4, Dectin-1, and mannose receptor MR were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a .",
"Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a . In vitro assay showed similar trends. As shown in Figure 5 b , Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model.",
"These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro.",
"The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza .",
"Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus including H1N1 and H3N2 subtypes . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro.",
"The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment 25 mg/kg body weight was significantly higher than in mice receiving PBS 50% to 0% . In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury.",
"In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect . However, it is unclear whether the lack of proinflammatory cytokines such as TNFand IL-6 facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections .",
"Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive .",
"Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage data not show . The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival .",
"The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival . TNF-has been reported to exert a defensive effect against influenza infection in vitro . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS.",
"In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response.",
"In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro.",
"In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection.",
"Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products."
] | 1,585 | 5,225 |
Which type of bacteria are implicated in carrying genes of drug resistance?
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Gammaproteobacteria
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[
"There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein GFP -expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas <30 in.",
"However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas <30 in. during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient.",
"We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient. IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks.",
"Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients. Text: D espite early reports . . . . .",
"Text: D espite early reports . . . . . , the premise that hand-wash sink traps can act as reservoirs of bacteria that cause nosocomial infections has been frequently overlooked. There has recently been an alarming increase in sink-related outbreaks worldwide, with many reports establishing an observational link . . . . . . . . . A sink often operates as an open conduit to wastewater in a patient care area that is often in the same room as the patient. Health care establishments often invest in desperate interventions to deal with nosocomial outbreaks. The preferred method for addressing most of the environmentrelated transmission is to employ enhanced cleaning using chemical and physical agents . .",
"The preferred method for addressing most of the environmentrelated transmission is to employ enhanced cleaning using chemical and physical agents . . Unfortunately, routine approaches are inefficient in completely eliminating drug-resistant Gammaproteobacteria in an inaccessible microbiologically active area such as a sink trap 6, . . . . . . The wet, humid, and relatively protected environment in a sink trap favors the formation of rich stable microbial communities . . These communities will be exposed to liquids and waste that are discarded in a sink and may include antimicrobials, discarded beverages, soap, presumably pathogenic bacteria from health care workers' hands, and other items.",
". These communities will be exposed to liquids and waste that are discarded in a sink and may include antimicrobials, discarded beverages, soap, presumably pathogenic bacteria from health care workers' hands, and other items. In short, sink traps could serve as a breeding ground for opportunistic and highly antimicrobial-resistant bacteria that cannot be easily cleaned or removed . . . . . . . There are many reports of a genetic association between pathogens found in sink traps and those found in patients . . However, surprisingly little work has been done to understand the microscale transmission dynamics.",
". However, surprisingly little work has been done to understand the microscale transmission dynamics. It was previously demonstrated using a suspension of fluorescent particles Glo Germ; Glo Germ Co., Moab, UT that material injected into the P-trap gets dispersed around a hand-washing sink . . This result however has not been replicated hitherto in the follow-up studies. Dispersion has never been investigated with living organisms.",
". This result however has not been replicated hitherto in the follow-up studies. Dispersion has never been investigated with living organisms. Ultimately, many details remain unaddressed surrounding the spread of Enterobacteriaceae in sink-trap wastewater systems: i can organisms grow retrograde from the P-trap water to the sink strainer, ii can organisms spread from one sink to another along the internal surfaces of pipes with shared drainage systems, and iii which portion of a colonized drain pipe results in dispersion into the sink bowl during a hand-washing event? We aim to better understand the dispersion dynamics of Gammaproteobacteria living in the wastewater of a sink strainer and P-trap into an area where patients and health care workers could be exposed. To study this dynamic, we used a surrogate organism that could be easily tracked while remaining in the Enterobacteriaceae family, where some of the most concerning threats in antimicrobial resistance are developing .",
"We aim to better understand the dispersion dynamics of Gammaproteobacteria living in the wastewater of a sink strainer and P-trap into an area where patients and health care workers could be exposed. To study this dynamic, we used a surrogate organism that could be easily tracked while remaining in the Enterobacteriaceae family, where some of the most concerning threats in antimicrobial resistance are developing . . Growth and colonization of GFP-expressing E. coli in the P-trap. In the first 14 days following the installation of the P-trap with established green fluorescent protein GFP -expressing Escherichia coli and just water running from the faucet, GFPexpressing E. coli was not detected in the tailpipe beyond 1.5 in. above the liquid level in the P-trap.",
"In the first 14 days following the installation of the P-trap with established green fluorescent protein GFP -expressing Escherichia coli and just water running from the faucet, GFPexpressing E. coli was not detected in the tailpipe beyond 1.5 in. above the liquid level in the P-trap. GFP-expressing E. coli, however, was found to be viable in the P-trap without any nutrients added. A nutrient regimen was then instituted to understand the influence of nutrients on mobility and upward growth. The addition of tryptic soy broth TSB promoted GFP-expressing E. coli growth as early as day 1, with growth observed in the tailpipe 2 in. above the liquid surface in the P-trap Table 1 .",
"The addition of tryptic soy broth TSB promoted GFP-expressing E. coli growth as early as day 1, with growth observed in the tailpipe 2 in. above the liquid surface in the P-trap Table 1 . On day 7, the strainer ϳ8 in. above the liquid in the P-trap was found to be colonized with GFP-expressing E. coli. This translates to an average growth rate of 1 in./day along the length of the tailpipe with the addition of nutrients and without faucet operation. GFP-expressing E. coli was not detected in the faucet water. Sink-to-sink transmission of bacteria.",
"GFP-expressing E. coli was not detected in the faucet water. Sink-to-sink transmission of bacteria. In these experiments, a flanking sink sink 5 was the only P-trap inoculated with GFP-expressing E. coli and therefore was the sole source for transmission to the connected sinks. Starting with a lower inoculum concentration 10 3 CFU/ml in sink 5, on day 7, GFP-expressing E. coli was detected in the sink 2 and sink 3 P-traps Fig. 1a . With inoculum concentrations of 10 6 CFU/ml and Ͼ10 10 CFU/ml in sink 5, all of the sink P-traps in the sink gallery with the exception of sink 1 were found to be colonized with GFP-expressing E. coli after 7 days Fig.",
"1a . With inoculum concentrations of 10 6 CFU/ml and Ͼ10 10 CFU/ml in sink 5, all of the sink P-traps in the sink gallery with the exception of sink 1 were found to be colonized with GFP-expressing E. coli after 7 days Fig. 1b and c . Faucet water and aerators tested negative for GFP-expressing E. coli. Irrespective of the starting inoculum concentration, on day 7 the highest level of colonization was recorded in the sink 3 P-trap. After day 7, when the nutrient regimen described previously was followed for an additional 7 days in each of the sinks in the sink gallery with an inoculum concentration of Ͼ10 10 CFU/ml, GFP-expressing E. coli was detected in the strainers of sinks 2 and 3 on day 14.",
"Irrespective of the starting inoculum concentration, on day 7 the highest level of colonization was recorded in the sink 3 P-trap. After day 7, when the nutrient regimen described previously was followed for an additional 7 days in each of the sinks in the sink gallery with an inoculum concentration of Ͼ10 10 CFU/ml, GFP-expressing E. coli was detected in the strainers of sinks 2 and 3 on day 14. This finding validated the upward growth and growth rate in the tailpipe when nutrients were added. Nonfluorescent colonies were occasionally observed in the P-trap water samples collected from the sinks, which were subsequently identified to be Pseudomonas sp. or Stenotrophomonas maltophilia, and fluorescent colonies were confirmed to be E. coli. Dispersion of microspheres from sinks.",
"or Stenotrophomonas maltophilia, and fluorescent colonies were confirmed to be E. coli. Dispersion of microspheres from sinks. In the first dispersion experiment, when fluorescent microspheres were inoculated into the offset drain tailpiece only 4 in. below the strainer, no microspheres were detected on the polyester sheets placed on the counter space. However, when the sink bowl was coated with the microspheres, polyester sheets overlaid on the counter space captured the dispersed microspheres caused by the faucet operation. Dispersion was observed on almost all zones of the sink counter space Fig. 2 .",
"Dispersion was observed on almost all zones of the sink counter space Fig. 2 . Relatively higher levels of dispersion were observed along the major and minor axes of the elliptical sink bowl zones 2, 5, 6, 9, 11, and 12 . Anterior corners of the sink counter space zones 4 and 7 , which were most distant from the impact of water in the sink bowl, received the lowest dispersion. Dispersion of GFP-expressing E. coli from sinks. Initially the P-trap alone was inoculated with GFP-expressing E. coli and carefully installed, keeping the tailpipe and strainer free of GFP-expressing E. coli before operating the faucets.",
"Dispersion of GFP-expressing E. coli from sinks. Initially the P-trap alone was inoculated with GFP-expressing E. coli and carefully installed, keeping the tailpipe and strainer free of GFP-expressing E. coli before operating the faucets. No fluorescent CFU were observed on the plates placed on the counter or attached to the bowl surface after faucet operation. Similarly, no fluorescent CFU were detected when GFPexpressing E. coli was inoculated into the offset drain tailpiece only 4 in. below the strainer. Interestingly, when there was conspicuous water backup over the strainer as a result of a higher water flow rate from the faucet than the drainage rate from the P-trap, dispersal was detected on the plates attached to the bowl surface.",
"below the strainer. Interestingly, when there was conspicuous water backup over the strainer as a result of a higher water flow rate from the faucet than the drainage rate from the P-trap, dispersal was detected on the plates attached to the bowl surface. The dispersion pattern recorded when the sink bowl was coated with GFPexpressing E. coli was comparable to the pattern recorded when the sink bowl was coated with fluorescent microspheres Fig. 2 . Dispersion was significantly higher along the axes zones 6, 9, 11, and 12 and lower at the corners of the sink counter space zones 4, 7, and 10 . In contrast, dispersion of GFP-expressing E. coli caused by the faucet operation was much more extensive when the strainer was allowed to be colonized with GFPexpressing E. coli prior to the dispersion experiment.",
"Dispersion was significantly higher along the axes zones 6, 9, 11, and 12 and lower at the corners of the sink counter space zones 4, 7, and 10 . In contrast, dispersion of GFP-expressing E. coli caused by the faucet operation was much more extensive when the strainer was allowed to be colonized with GFPexpressing E. coli prior to the dispersion experiment. In addition to the sink counter space, we measured dispersion to the sink bowl, faucet, faucet handles, splatter shields, and the extended counter surface. Dispersion of GFP-expressing E. coli was highest on the plates attached to the sink bowl Fig. 3b . Further, dispersion was greater along the Ϫ\" and \"ϩ\" denote the absence and presence of GFP-expressing E. coli, respectively.",
"3b . Further, dispersion was greater along the Ϫ\" and \"ϩ\" denote the absence and presence of GFP-expressing E. coli, respectively. minor axis of the sink bowl Fig. 3b , zones B3, B4, and B10 than along the major axis of the sink bowl, associated with a shorter distance from the strike point of the faucet water to the bowl along this axis. The next highest CFU count from the dispersal was recorded on the counter area near the faucets Fig. 3a , zones 12 and 11 . A similar pattern of higher dispersion near the faucets and lower dispersion at the corners of the counter space Fig.",
"3a , zones 12 and 11 . A similar pattern of higher dispersion near the faucets and lower dispersion at the corners of the counter space Fig. 3a , zones 4, 7, and 10 was also observed using microspheres. Dispersion was also recorded in other zones of the counter space, on the Plexiglas splatter shields, faucets, faucet handles, and extended surface Fig. 3c . There were no GFP-expressing E. coli CFU recorded on plates placed beyond 30 in. from the strainer, demarcating the range of dispersion under these experimental conditions. Table 2 gives a summary of the total distribution loads recorded using fluorescent microspheres and GFP-expressing E. coli across each experiment.",
"from the strainer, demarcating the range of dispersion under these experimental conditions. Table 2 gives a summary of the total distribution loads recorded using fluorescent microspheres and GFP-expressing E. coli across each experiment. The loads of dispersion on the sink counter were comparable when the sink bowl was coated with microspheres or GFP-expressing E. coli before the faucet operation. Although the dispersion load on the sink counter was lower when the sink strainer was colonized, it is interesting to note that the sink bowl received the highest dispersion. To mimic dispersion in a hospital setting, we first investigated whether GFPexpressing E. coli would establish consistent colonization in a sink trap as many other Gammaproteobacteria implicated in nosocomial outbreaks have done . .",
"To mimic dispersion in a hospital setting, we first investigated whether GFPexpressing E. coli would establish consistent colonization in a sink trap as many other Gammaproteobacteria implicated in nosocomial outbreaks have done . . Many recent reports demonstrate that P-traps become colonized with highly consequential Gammaproteobacteria, which then results in nosocomial transmission . . The retained water in a sink P-trap is present to provide a water barrier to prevent off-gassing of sewer smell, but it may inadvertently provide favorable conditions for pathogenic and opportunistic antibiotic-resistant microorganisms to survive and develop resilient biofilms . . However, the mechanism of dispersal of the bacteria in the P-trap to patients or the surrounding health care area had not been fully elucidated.",
". However, the mechanism of dispersal of the bacteria in the P-trap to patients or the surrounding health care area had not been fully elucidated. We began with the hypothesis that the bacteria originate from the P-trap via droplet creation when the water from the faucet hits the P-trap water, thus contaminating the sink bowl and the surrounding area. The finding supporting this theory had been previously reported using Glo Germ particles . . However, in the present study with careful attention to avoid strainer and tail piece contamination, the dispersal directly from the sink P-trap with either microspheres or GFP-expressing E. coli could not be reproduced as previously reported . .",
"However, in the present study with careful attention to avoid strainer and tail piece contamination, the dispersal directly from the sink P-trap with either microspheres or GFP-expressing E. coli could not be reproduced as previously reported . . Rather this work demonstrates a different, more staged mode of transmission from a P-trap reservoir to the sink and surrounding environment. GFP-expressing E. coli in the P-trap alone was sustained for 14 days but did not grow or mobilize up the tailpipe to the strainer with just intermittent water exposure. However, when nutrients were subsequently added to the system, the organisms rapidly grew up the tailpipe to the strainer at approximately an inch per day. In a real-world setting, motility of bacteria inside the tailpipe is restricted to relatively sporadic and brief wetting events in which swimming is an opportunity to colonize new surfaces.",
"However, when nutrients were subsequently added to the system, the organisms rapidly grew up the tailpipe to the strainer at approximately an inch per day. In a real-world setting, motility of bacteria inside the tailpipe is restricted to relatively sporadic and brief wetting events in which swimming is an opportunity to colonize new surfaces. It is assumed that once established, the biofilm promotes the upward growth of GFP-expressing E. coli in the tailpipe at an accelerated rate. The nutrient regimen that promoted colonization in our model reflects our and others' observations of items commonly disposed of in hospital sinks intravenous fluids, feeding supplements, and leftover beverages . . Transmission of bacteria between sinks via a common pipe was a key finding in this study as this highlights the concept that premise plumbing may be a more continuous system with shared microbiology than a single isolated sink.",
". Transmission of bacteria between sinks via a common pipe was a key finding in this study as this highlights the concept that premise plumbing may be a more continuous system with shared microbiology than a single isolated sink. The sink gallery used in this study provided a unique in situ advantage to investigate sink-to-sink transmission of bacteria through common drains. The two possible mechanisms for P-trap strainers becoming colonized are seeding of organisms from above and retrograde spread of organisms along common pipes in a hospital wastewater infrastructure. Here we demonstrate that it is possible for GFP-expressing E. coli to contaminate adjacent P-traps with just time and water given a standard U.S. code piping rise of 0.25 ft/ft. Sink-to-sink or retrograde transmission may explain the recurrence of pathogen colonization following intervention strategies like disinfection or replacement of plumbing .",
"Here we demonstrate that it is possible for GFP-expressing E. coli to contaminate adjacent P-traps with just time and water given a standard U.S. code piping rise of 0.25 ft/ft. Sink-to-sink or retrograde transmission may explain the recurrence of pathogen colonization following intervention strategies like disinfection or replacement of plumbing . . Sink 3 was lowest on the slope in the drain line see Fig. 4 with arguably the most opportunity for reflux and retrograde wetting. Sink 1, on the other hand, was farthest away from the source sink 5 , and its P-trap had the greatest incline in the drain line connecting the sinks, which could perhaps contribute to the reasons there was no GFP-expressing E. coli colonization detected in it after 7 days.",
"4 with arguably the most opportunity for reflux and retrograde wetting. Sink 1, on the other hand, was farthest away from the source sink 5 , and its P-trap had the greatest incline in the drain line connecting the sinks, which could perhaps contribute to the reasons there was no GFP-expressing E. coli colonization detected in it after 7 days. There has been more investigation about microbiologic dynamics of infectious viral particles such as those of severe acute respiratory syndrome SARS and Ebola viruses through premise plumbing systems . . . . However, the microbiology, sustainability, and dynamics might be very different, although the backflow and inoculation issues could have some parallels when comparing viruses to bacteria.",
". . However, the microbiology, sustainability, and dynamics might be very different, although the backflow and inoculation issues could have some parallels when comparing viruses to bacteria. As Enterobacteriaceae can either multiply or remain viable for long periods of time in biofilms coating the interior of P-traps and the connected plumbing, it may not be sustainable to target any intervention limited to a single isolated sink as a source of a particular pathogen. Data from different dispersal experiments suggest that although P-traps can act as the source or the reservoir of pathogens, the physical presence of the organism in the sink bowl or colonization of the strainer is necessary for the dispersal to occur. Colonization of strainers or drains reported in earlier studies .",
"Data from different dispersal experiments suggest that although P-traps can act as the source or the reservoir of pathogens, the physical presence of the organism in the sink bowl or colonization of the strainer is necessary for the dispersal to occur. Colonization of strainers or drains reported in earlier studies . was perhaps a result of ascending biofilm growth from the P-trap to the strainer or introduction through contaminated fluids. Many of the studies used swab samples, which likely sampled the strainer rather than P-trap water . . Once the strainer was colonized, the water from the faucet resulted in GFP-expressing E. coli dispersion in the bowl and to the surrounding surfaces of up to 30 in.",
". Once the strainer was colonized, the water from the faucet resulted in GFP-expressing E. coli dispersion in the bowl and to the surrounding surfaces of up to 30 in. The range of dispersal . . Greater dispersal near the faucet may be attributed to the specific designs of the sink bowl and faucet in this study, which determine the contact angle of water impact. This is an important finding since many sinks in hospitals are similar in design, with faucet handles representing a high-touch surface for the sink users . . It can also be concluded from the dispersion experiments that secondary and successive dispersals would likely increase the degree and the scope of dispersion.",
". It can also be concluded from the dispersion experiments that secondary and successive dispersals would likely increase the degree and the scope of dispersion. There are several limitations to this work. First the use of similar sink bowls across these sinks only examines the dispersion pattern of this particular sink design. Similarly the sink-to-sink transmission may not be applicable to all wastewater plumbing systems as the fixtures on the pipe are very close together, unlike most layouts in health care settings. However, we speculate that transmission could occur on larger systems over greater time scales, especially if heavy nutrient and contamination loads were also included.",
"Similarly the sink-to-sink transmission may not be applicable to all wastewater plumbing systems as the fixtures on the pipe are very close together, unlike most layouts in health care settings. However, we speculate that transmission could occur on larger systems over greater time scales, especially if heavy nutrient and contamination loads were also included. GFP-expressing E. coli is a laboratory surrogate, and the putative biofilms established in the short time frame of our experiments are unlikely to be as complex or stable as biofilms developed in a hospital wastewater system over many years. However, to address the monomicrobial dominance of the GFP-expressing E. coli added to the system, we kept the system open, and other environmental organisms were able to cocolonize in an attempt to mimic the hospital system. Another limitation was the need to add nutrients to the drain to ensure rapid and robust colonization. We are not clear how widespread the practice of disposing of dextrose-containing intravenous fluids or leftover beverages in the hand-wash sinks is; however, we have observed this practice, and anecdotally it appears to be relatively common in the United States.",
"Another limitation was the need to add nutrients to the drain to ensure rapid and robust colonization. We are not clear how widespread the practice of disposing of dextrose-containing intravenous fluids or leftover beverages in the hand-wash sinks is; however, we have observed this practice, and anecdotally it appears to be relatively common in the United States. We also did not completely characterize the droplet sizes, nor do we demonstrate air sampling to understand if the dispersion is only droplet or if there are also aerosols that contain GFP-expressing E. coli. This would require additional testing and is planned as future work. In summary, this work for the first time better models the mechanisms of spread of multidrug-resistant pathogens arising from the sink drain and infecting patients. Droplet dispersion from the P-trap does not happen directly.",
"In summary, this work for the first time better models the mechanisms of spread of multidrug-resistant pathogens arising from the sink drain and infecting patients. Droplet dispersion from the P-trap does not happen directly. Rather it is a multistage process: dispersal originates from the strainer and/or the bowl after growth of the biofilm up from the microbial reservoir of the P-trap. We also demonstrate sink-to-sink transmission via a common sanitary pipe. This work could have implications for patient safety, infection control, and interventions as well as the design of future hospital plumbing systems to eliminate this mode of transmission to vulnerable hospitalized patients. Sink gallery design.",
"This work could have implications for patient safety, infection control, and interventions as well as the design of future hospital plumbing systems to eliminate this mode of transmission to vulnerable hospitalized patients. Sink gallery design. A dedicated sink gallery was set up to simulate hospital hand-washing sinks. The gallery was comprised of five sink modules assembled next to each other Fig. 4 . The five hand-wash sink stations were identical in bowl designs and dimensions and were modeled from the most common intensive care unit hand-washing sink type in the acute care hospital at the University of Virginia Medical Center. Partitions made of 24-in.-high Plexiglas sheet were installed between the sinks to prevent splatter and cross contamination.",
"The five hand-wash sink stations were identical in bowl designs and dimensions and were modeled from the most common intensive care unit hand-washing sink type in the acute care hospital at the University of Virginia Medical Center. Partitions made of 24-in.-high Plexiglas sheet were installed between the sinks to prevent splatter and cross contamination. Each sink module was built with Corian integrated sink/countertops without an overflow and fitted with an 8-in. centerset 2-handle gooseneck faucet Elkay, Oak Brook, IL . The drain line Dearborn Brass-Oatey, Cleveland, OH . All of the fixtures were made of brass with chrome plating. Each of the sink P-traps was connected to a 3-in.",
"All of the fixtures were made of brass with chrome plating. Each of the sink P-traps was connected to a 3-in. common cast-iron pipe sloping into a T-joint leading into the building sanitary line located behind sink 3 Fig. 4 . Inoculation, growth, and establishment of GFP-expressing E. coli in sink P-traps. For the GFP-expressing E. coli strain ATCC 25922GFP , the green fluorescent protein GFP gene is contained on a plasmid that also contains an ampicillin resistance gene. A single isolated colony of GFP-expressing E. coli grown from a Ϫ80°C stock was inoculated into 5 ml tryptic soy broth TSB Becton, Dickinson and Company, Sparks, MD containing 100 g/ml ampicillin ATCC medium 2855 .",
"For the GFP-expressing E. coli strain ATCC 25922GFP , the green fluorescent protein GFP gene is contained on a plasmid that also contains an ampicillin resistance gene. A single isolated colony of GFP-expressing E. coli grown from a Ϫ80°C stock was inoculated into 5 ml tryptic soy broth TSB Becton, Dickinson and Company, Sparks, MD containing 100 g/ml ampicillin ATCC medium 2855 . The inoculum concentration and method varied for each experiment. For establishment of GFP-expressing E. coli in sink P-traps, new autoclaved P-traps were filled with 100 ml 0.1ϫ TSB and inoculated with ϳ10 3 CFU/ml GFPexpressing E. coli. Following inoculation, both ends of the P-traps were covered with perforated Parafilm Bemis, Inc., Oshkosh, WI and allowed to incubate at room temperature 22 Ϯ 2°C for 14 days to facilitate adherent bacterial growth. The medium in the P-trap was decanted and replaced with fresh 0.1ϫ TSB every 48 h. An aliquot of decanted medium and a swab sample from the inner surface of the P-trap were plated on tryptic soy agar Becton, Dickinson and Company, Sparks, MD plates containing 100 g/ml ampicillin TSA to monitor the growth of GFP-expressing E. coli in the P-traps.",
"Following inoculation, both ends of the P-traps were covered with perforated Parafilm Bemis, Inc., Oshkosh, WI and allowed to incubate at room temperature 22 Ϯ 2°C for 14 days to facilitate adherent bacterial growth. The medium in the P-trap was decanted and replaced with fresh 0.1ϫ TSB every 48 h. An aliquot of decanted medium and a swab sample from the inner surface of the P-trap were plated on tryptic soy agar Becton, Dickinson and Company, Sparks, MD plates containing 100 g/ml ampicillin TSA to monitor the growth of GFP-expressing E. coli in the P-traps. TSA plates were incubated overnight at 37°C, and CFU fluorescing under UV light were enumerated. All preparatory culturing of GFP-expressing E. coli took place in a separate room from the sink gallery to avoid unintentional contamination. Installation of P-traps colonized with GFP-expressing E.coli. After the 14-day incubation, P-traps were fastened into the plumbing of the sinks Fig.",
"Installation of P-traps colonized with GFP-expressing E.coli. After the 14-day incubation, P-traps were fastened into the plumbing of the sinks Fig. 5a . The remainder of the drain line was either autoclaved strainer, tailpipe, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI , maintaining at least 1 min of contact time. After the P-trap was installed, a daily regimen was followed in which 25 ml of TSB followed by 25 ml of 0.9% NaCl solution saline were added in the ratio 1:3 via the strainer Fig. 5b to mimic the potential nutrient exposure in the hospital.",
"After the P-trap was installed, a daily regimen was followed in which 25 ml of TSB followed by 25 ml of 0.9% NaCl solution saline were added in the ratio 1:3 via the strainer Fig. 5b to mimic the potential nutrient exposure in the hospital. Sampling and enumeration of GFP-expressing E. coli. To monitor the growth of GFP-expressing E. coli in the plumbing, sampling ports were drilled along the length of the tailpiece between the P-trap and the strainer and the trap arm between the P-trap and the common line . These holes were fitted with size 00 silicone stoppers Cole-Parmer, Vernon Hills, IL Fig. 5a .",
"These holes were fitted with size 00 silicone stoppers Cole-Parmer, Vernon Hills, IL Fig. 5a . Sterile cotton swabs Covidien, Mansfield, MA presoaked in saline were inserted through these sampling ports, and samples were collected by turning the swab in a circular motion on the inner surface ϳ20 cm 2 of the tailpipes. Sample swabs were pulse-vortexed in 3 ml saline, and serial dilutions were plated on TSA. The strainer, faucet aerator, and bowl surface were sampled with presoaked swabs and processed as described earlier. Sink-to-sink transmission of bacteria. To investigate sink-to-sink transmission of bacteria, a distal sink sink 5 Fig.",
"Sink-to-sink transmission of bacteria. To investigate sink-to-sink transmission of bacteria, a distal sink sink 5 Fig. 4 was fitted with a P-trap inoculated with GFP-expressing E. coli. The effects of different inoculum concentrations of GFP-expressing E. coli-10 3 , 10 6 , and Ͼ10 10 CFU/ml colonized for 14 days -were investigated. Identification to the species level of fluorescent and nonfluorescent colonies identified from mixed pipe cultures was performed using a matrix-assisted laser desorption-ionization MALDI -time of flight MALDI-TOF mass spectrometer Vitek-MS; bioMérieux, Durham, NC . The wastewater paths of sinks 1 to 4 were either autoclaved strainer, tailpipe, P-traps, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI .",
"Identification to the species level of fluorescent and nonfluorescent colonies identified from mixed pipe cultures was performed using a matrix-assisted laser desorption-ionization MALDI -time of flight MALDI-TOF mass spectrometer Vitek-MS; bioMérieux, Durham, NC . The wastewater paths of sinks 1 to 4 were either autoclaved strainer, tailpipe, P-traps, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI . Faucets on each of the five sinks were turned on simultaneously for 1 min, supplying water at a flow rate of 8 liters/min, once every 24 h for 7 days. No additional feed to any of the sinks was added during this period of 7 days. On days 0 and 7, P-traps on each of the five sinks were unfastened, and swab samples from the P-trap were collected and processed as described earlier. Dispersion measured using fluorescent microspheres.",
"On days 0 and 7, P-traps on each of the five sinks were unfastened, and swab samples from the P-trap were collected and processed as described earlier. Dispersion measured using fluorescent microspheres. Fluoresbrite YO carboxylate microspheres Polysciences, Inc. which had a 1-m diameter and maximum excitation and emission of 529 nm and 546 nm, respectively, were used as a tracer in the preliminary experiments to understand droplet dispersion from the hand-wash sinks. To test whether microspheres could be dispersed from below the sink strainer, a 1-ml suspension of microspheres ϳ10 10 particles was injected through a strainer attached to a Hert 4.5-in. offset drain tailpiece typically used for wheelchair-accessible sinks American Standard, model 7723018.002 Fig. 5c .",
"offset drain tailpiece typically used for wheelchair-accessible sinks American Standard, model 7723018.002 Fig. 5c . The vertical distance between the strainer and microsphere suspension injected into the tailpipe was ϳ4 in. Counter space around the sink bowl was thoroughly wiped with alcohol wipes Covidien Webcol 6818; Kendall , and polyester sheets precut to appropriate shapes were placed on the counter to cover the entire sink counter and labeled according to position Fig. 6a . The faucet was turned on for 5 min at a water flow rate of 1.8 to 3.0 liters/min.",
"6a . The faucet was turned on for 5 min at a water flow rate of 1.8 to 3.0 liters/min. Polyester sheets were harvested and immediately analyzed using a ChemiDoc MP system Bio-Rad Laboratories, Inc. with an exposure time of 5 s. Fluorescent microspheres were enumerated from the digital micrographs using the Image Lab Software Bio-Rad Laboratories, Inc. . To test whether microspheres could be dispersed from the surface of the sink bowl, the sink bowl was evenly coated with a 20-ml microsphere suspension ϳ10 10 particles/ml using a disposable swab Sage Products, Inc., Cary, IL , and the dispersion experiment was repeated following the protocol described above. To ascertain there was no nonspecific background fluorescence in the sink and/or the water from the faucet, a control using the same protocol but without the fluorescent microspheres was performed before each experiment. Dispersion measured using GFP-expressing E.coli.",
"To ascertain there was no nonspecific background fluorescence in the sink and/or the water from the faucet, a control using the same protocol but without the fluorescent microspheres was performed before each experiment. Dispersion measured using GFP-expressing E.coli. Dispersion using GFP-expressing E. coli was investigated in three experiments. To test whether live organisms in the P-trap could be dispersed by running water, ϳ10 10 CFU/ml GFP-expressing E. coli in saline was added to an autoclaved P-trap and fitted into the drain line that was preautoclaved strainer, tailpipe, and trap arms . Similarly, to test whether live organisms could be dispersed from the tailpieces of wheelchair-accessible sinks, a suspension of ϳ10 10 CFU/ml GFP-expressing E. coli was added with a syringe through the strainer into the Hert 4.5-ft offset drain tailpiece Fig. 5c .",
"Similarly, to test whether live organisms could be dispersed from the tailpieces of wheelchair-accessible sinks, a suspension of ϳ10 10 CFU/ml GFP-expressing E. coli was added with a syringe through the strainer into the Hert 4.5-ft offset drain tailpiece Fig. 5c . Just as in the microsphere dispersion experiment, the vertical distance between the strainer and GFP-expressing E. coli suspension injected into the tailpipe was ϳ4 in. We next tested whether live organisms from the surface of the sink bowl could be dispersed by running water. The sink bowl surface was evenly coated with an approximately 20-ml suspension of 10 10 CFU/ml GFP-expressing E. coli. Finally, to mimic all of these conditions, a P-trap colonized with GFP-expressing E. coli for 14 days was installed, and a nutrient regimen Fig.",
"The sink bowl surface was evenly coated with an approximately 20-ml suspension of 10 10 CFU/ml GFP-expressing E. coli. Finally, to mimic all of these conditions, a P-trap colonized with GFP-expressing E. coli for 14 days was installed, and a nutrient regimen Fig. 5b was followed for 14 days to intentionally promote the GFP-expressing E. coli colonization in the attached tailpipe and strainer. On day 15, the dispersion experiment was performed. Before each of the GFP-expressing E. coli dispersion experiments, the counter space was thoroughly disinfected with Caviwipes-1. TSA plates were then positioned on the sink counter surrounding the bowl and an extension platform Fig. 6b .",
"TSA plates were then positioned on the sink counter surrounding the bowl and an extension platform Fig. 6b . Additional plates were attached to the sink bowl, faucets, Plexiglas partitions, and faucet handles using adhesive tape. TSA plates were also placed 3 m away from the sink as negative controls. The faucet was turned on for 5 min with a water flow rate of 1.8 to 3.0 liters/min. Lids of the TSA plates were removed only during faucet operation. Swab samples from the faucet aerators before and after operation were collected and plated on TSA.",
"Lids of the TSA plates were removed only during faucet operation. Swab samples from the faucet aerators before and after operation were collected and plated on TSA. Prior to each dispersion experiment, 50 ml water from the faucet was also collected, and aliquots were plated to assess for the presence of GFP-expressing E. coli in source water and ensure cross contamination of GFP-expressing E. coli had not occurred. A control dispersion experiment was also performed using the same protocol prior to GFP-expressing E. coli inoculation in each case. Dispersion per defined area CFU per square centimeter was deduced by dividing the CFU counts in the TSA plate by the surface area of the TSA plate."
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What may be a likely cause of sink-to-sink spreading of pathogens in the hospital setting?
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via a common sanitary pipe
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[
"There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein GFP -expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas <30 in.",
"However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas <30 in. during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient.",
"We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient. IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks.",
"Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients. Text: D espite early reports . . . . .",
"Text: D espite early reports . . . . . , the premise that hand-wash sink traps can act as reservoirs of bacteria that cause nosocomial infections has been frequently overlooked. There has recently been an alarming increase in sink-related outbreaks worldwide, with many reports establishing an observational link . . . . . . . . . A sink often operates as an open conduit to wastewater in a patient care area that is often in the same room as the patient. Health care establishments often invest in desperate interventions to deal with nosocomial outbreaks. The preferred method for addressing most of the environmentrelated transmission is to employ enhanced cleaning using chemical and physical agents . .",
"The preferred method for addressing most of the environmentrelated transmission is to employ enhanced cleaning using chemical and physical agents . . Unfortunately, routine approaches are inefficient in completely eliminating drug-resistant Gammaproteobacteria in an inaccessible microbiologically active area such as a sink trap 6, . . . . . . The wet, humid, and relatively protected environment in a sink trap favors the formation of rich stable microbial communities . . These communities will be exposed to liquids and waste that are discarded in a sink and may include antimicrobials, discarded beverages, soap, presumably pathogenic bacteria from health care workers' hands, and other items.",
". These communities will be exposed to liquids and waste that are discarded in a sink and may include antimicrobials, discarded beverages, soap, presumably pathogenic bacteria from health care workers' hands, and other items. In short, sink traps could serve as a breeding ground for opportunistic and highly antimicrobial-resistant bacteria that cannot be easily cleaned or removed . . . . . . . There are many reports of a genetic association between pathogens found in sink traps and those found in patients . . However, surprisingly little work has been done to understand the microscale transmission dynamics.",
". However, surprisingly little work has been done to understand the microscale transmission dynamics. It was previously demonstrated using a suspension of fluorescent particles Glo Germ; Glo Germ Co., Moab, UT that material injected into the P-trap gets dispersed around a hand-washing sink . . This result however has not been replicated hitherto in the follow-up studies. Dispersion has never been investigated with living organisms.",
". This result however has not been replicated hitherto in the follow-up studies. Dispersion has never been investigated with living organisms. Ultimately, many details remain unaddressed surrounding the spread of Enterobacteriaceae in sink-trap wastewater systems: i can organisms grow retrograde from the P-trap water to the sink strainer, ii can organisms spread from one sink to another along the internal surfaces of pipes with shared drainage systems, and iii which portion of a colonized drain pipe results in dispersion into the sink bowl during a hand-washing event? We aim to better understand the dispersion dynamics of Gammaproteobacteria living in the wastewater of a sink strainer and P-trap into an area where patients and health care workers could be exposed. To study this dynamic, we used a surrogate organism that could be easily tracked while remaining in the Enterobacteriaceae family, where some of the most concerning threats in antimicrobial resistance are developing .",
"We aim to better understand the dispersion dynamics of Gammaproteobacteria living in the wastewater of a sink strainer and P-trap into an area where patients and health care workers could be exposed. To study this dynamic, we used a surrogate organism that could be easily tracked while remaining in the Enterobacteriaceae family, where some of the most concerning threats in antimicrobial resistance are developing . . Growth and colonization of GFP-expressing E. coli in the P-trap. In the first 14 days following the installation of the P-trap with established green fluorescent protein GFP -expressing Escherichia coli and just water running from the faucet, GFPexpressing E. coli was not detected in the tailpipe beyond 1.5 in. above the liquid level in the P-trap.",
"In the first 14 days following the installation of the P-trap with established green fluorescent protein GFP -expressing Escherichia coli and just water running from the faucet, GFPexpressing E. coli was not detected in the tailpipe beyond 1.5 in. above the liquid level in the P-trap. GFP-expressing E. coli, however, was found to be viable in the P-trap without any nutrients added. A nutrient regimen was then instituted to understand the influence of nutrients on mobility and upward growth. The addition of tryptic soy broth TSB promoted GFP-expressing E. coli growth as early as day 1, with growth observed in the tailpipe 2 in. above the liquid surface in the P-trap Table 1 .",
"The addition of tryptic soy broth TSB promoted GFP-expressing E. coli growth as early as day 1, with growth observed in the tailpipe 2 in. above the liquid surface in the P-trap Table 1 . On day 7, the strainer ϳ8 in. above the liquid in the P-trap was found to be colonized with GFP-expressing E. coli. This translates to an average growth rate of 1 in./day along the length of the tailpipe with the addition of nutrients and without faucet operation. GFP-expressing E. coli was not detected in the faucet water. Sink-to-sink transmission of bacteria.",
"GFP-expressing E. coli was not detected in the faucet water. Sink-to-sink transmission of bacteria. In these experiments, a flanking sink sink 5 was the only P-trap inoculated with GFP-expressing E. coli and therefore was the sole source for transmission to the connected sinks. Starting with a lower inoculum concentration 10 3 CFU/ml in sink 5, on day 7, GFP-expressing E. coli was detected in the sink 2 and sink 3 P-traps Fig. 1a . With inoculum concentrations of 10 6 CFU/ml and Ͼ10 10 CFU/ml in sink 5, all of the sink P-traps in the sink gallery with the exception of sink 1 were found to be colonized with GFP-expressing E. coli after 7 days Fig.",
"1a . With inoculum concentrations of 10 6 CFU/ml and Ͼ10 10 CFU/ml in sink 5, all of the sink P-traps in the sink gallery with the exception of sink 1 were found to be colonized with GFP-expressing E. coli after 7 days Fig. 1b and c . Faucet water and aerators tested negative for GFP-expressing E. coli. Irrespective of the starting inoculum concentration, on day 7 the highest level of colonization was recorded in the sink 3 P-trap. After day 7, when the nutrient regimen described previously was followed for an additional 7 days in each of the sinks in the sink gallery with an inoculum concentration of Ͼ10 10 CFU/ml, GFP-expressing E. coli was detected in the strainers of sinks 2 and 3 on day 14.",
"Irrespective of the starting inoculum concentration, on day 7 the highest level of colonization was recorded in the sink 3 P-trap. After day 7, when the nutrient regimen described previously was followed for an additional 7 days in each of the sinks in the sink gallery with an inoculum concentration of Ͼ10 10 CFU/ml, GFP-expressing E. coli was detected in the strainers of sinks 2 and 3 on day 14. This finding validated the upward growth and growth rate in the tailpipe when nutrients were added. Nonfluorescent colonies were occasionally observed in the P-trap water samples collected from the sinks, which were subsequently identified to be Pseudomonas sp. or Stenotrophomonas maltophilia, and fluorescent colonies were confirmed to be E. coli. Dispersion of microspheres from sinks.",
"or Stenotrophomonas maltophilia, and fluorescent colonies were confirmed to be E. coli. Dispersion of microspheres from sinks. In the first dispersion experiment, when fluorescent microspheres were inoculated into the offset drain tailpiece only 4 in. below the strainer, no microspheres were detected on the polyester sheets placed on the counter space. However, when the sink bowl was coated with the microspheres, polyester sheets overlaid on the counter space captured the dispersed microspheres caused by the faucet operation. Dispersion was observed on almost all zones of the sink counter space Fig. 2 .",
"Dispersion was observed on almost all zones of the sink counter space Fig. 2 . Relatively higher levels of dispersion were observed along the major and minor axes of the elliptical sink bowl zones 2, 5, 6, 9, 11, and 12 . Anterior corners of the sink counter space zones 4 and 7 , which were most distant from the impact of water in the sink bowl, received the lowest dispersion. Dispersion of GFP-expressing E. coli from sinks. Initially the P-trap alone was inoculated with GFP-expressing E. coli and carefully installed, keeping the tailpipe and strainer free of GFP-expressing E. coli before operating the faucets.",
"Dispersion of GFP-expressing E. coli from sinks. Initially the P-trap alone was inoculated with GFP-expressing E. coli and carefully installed, keeping the tailpipe and strainer free of GFP-expressing E. coli before operating the faucets. No fluorescent CFU were observed on the plates placed on the counter or attached to the bowl surface after faucet operation. Similarly, no fluorescent CFU were detected when GFPexpressing E. coli was inoculated into the offset drain tailpiece only 4 in. below the strainer. Interestingly, when there was conspicuous water backup over the strainer as a result of a higher water flow rate from the faucet than the drainage rate from the P-trap, dispersal was detected on the plates attached to the bowl surface.",
"below the strainer. Interestingly, when there was conspicuous water backup over the strainer as a result of a higher water flow rate from the faucet than the drainage rate from the P-trap, dispersal was detected on the plates attached to the bowl surface. The dispersion pattern recorded when the sink bowl was coated with GFPexpressing E. coli was comparable to the pattern recorded when the sink bowl was coated with fluorescent microspheres Fig. 2 . Dispersion was significantly higher along the axes zones 6, 9, 11, and 12 and lower at the corners of the sink counter space zones 4, 7, and 10 . In contrast, dispersion of GFP-expressing E. coli caused by the faucet operation was much more extensive when the strainer was allowed to be colonized with GFPexpressing E. coli prior to the dispersion experiment.",
"Dispersion was significantly higher along the axes zones 6, 9, 11, and 12 and lower at the corners of the sink counter space zones 4, 7, and 10 . In contrast, dispersion of GFP-expressing E. coli caused by the faucet operation was much more extensive when the strainer was allowed to be colonized with GFPexpressing E. coli prior to the dispersion experiment. In addition to the sink counter space, we measured dispersion to the sink bowl, faucet, faucet handles, splatter shields, and the extended counter surface. Dispersion of GFP-expressing E. coli was highest on the plates attached to the sink bowl Fig. 3b . Further, dispersion was greater along the Ϫ\" and \"ϩ\" denote the absence and presence of GFP-expressing E. coli, respectively.",
"3b . Further, dispersion was greater along the Ϫ\" and \"ϩ\" denote the absence and presence of GFP-expressing E. coli, respectively. minor axis of the sink bowl Fig. 3b , zones B3, B4, and B10 than along the major axis of the sink bowl, associated with a shorter distance from the strike point of the faucet water to the bowl along this axis. The next highest CFU count from the dispersal was recorded on the counter area near the faucets Fig. 3a , zones 12 and 11 . A similar pattern of higher dispersion near the faucets and lower dispersion at the corners of the counter space Fig.",
"3a , zones 12 and 11 . A similar pattern of higher dispersion near the faucets and lower dispersion at the corners of the counter space Fig. 3a , zones 4, 7, and 10 was also observed using microspheres. Dispersion was also recorded in other zones of the counter space, on the Plexiglas splatter shields, faucets, faucet handles, and extended surface Fig. 3c . There were no GFP-expressing E. coli CFU recorded on plates placed beyond 30 in. from the strainer, demarcating the range of dispersion under these experimental conditions. Table 2 gives a summary of the total distribution loads recorded using fluorescent microspheres and GFP-expressing E. coli across each experiment.",
"from the strainer, demarcating the range of dispersion under these experimental conditions. Table 2 gives a summary of the total distribution loads recorded using fluorescent microspheres and GFP-expressing E. coli across each experiment. The loads of dispersion on the sink counter were comparable when the sink bowl was coated with microspheres or GFP-expressing E. coli before the faucet operation. Although the dispersion load on the sink counter was lower when the sink strainer was colonized, it is interesting to note that the sink bowl received the highest dispersion. To mimic dispersion in a hospital setting, we first investigated whether GFPexpressing E. coli would establish consistent colonization in a sink trap as many other Gammaproteobacteria implicated in nosocomial outbreaks have done . .",
"To mimic dispersion in a hospital setting, we first investigated whether GFPexpressing E. coli would establish consistent colonization in a sink trap as many other Gammaproteobacteria implicated in nosocomial outbreaks have done . . Many recent reports demonstrate that P-traps become colonized with highly consequential Gammaproteobacteria, which then results in nosocomial transmission . . The retained water in a sink P-trap is present to provide a water barrier to prevent off-gassing of sewer smell, but it may inadvertently provide favorable conditions for pathogenic and opportunistic antibiotic-resistant microorganisms to survive and develop resilient biofilms . . However, the mechanism of dispersal of the bacteria in the P-trap to patients or the surrounding health care area had not been fully elucidated.",
". However, the mechanism of dispersal of the bacteria in the P-trap to patients or the surrounding health care area had not been fully elucidated. We began with the hypothesis that the bacteria originate from the P-trap via droplet creation when the water from the faucet hits the P-trap water, thus contaminating the sink bowl and the surrounding area. The finding supporting this theory had been previously reported using Glo Germ particles . . However, in the present study with careful attention to avoid strainer and tail piece contamination, the dispersal directly from the sink P-trap with either microspheres or GFP-expressing E. coli could not be reproduced as previously reported . .",
"However, in the present study with careful attention to avoid strainer and tail piece contamination, the dispersal directly from the sink P-trap with either microspheres or GFP-expressing E. coli could not be reproduced as previously reported . . Rather this work demonstrates a different, more staged mode of transmission from a P-trap reservoir to the sink and surrounding environment. GFP-expressing E. coli in the P-trap alone was sustained for 14 days but did not grow or mobilize up the tailpipe to the strainer with just intermittent water exposure. However, when nutrients were subsequently added to the system, the organisms rapidly grew up the tailpipe to the strainer at approximately an inch per day. In a real-world setting, motility of bacteria inside the tailpipe is restricted to relatively sporadic and brief wetting events in which swimming is an opportunity to colonize new surfaces.",
"However, when nutrients were subsequently added to the system, the organisms rapidly grew up the tailpipe to the strainer at approximately an inch per day. In a real-world setting, motility of bacteria inside the tailpipe is restricted to relatively sporadic and brief wetting events in which swimming is an opportunity to colonize new surfaces. It is assumed that once established, the biofilm promotes the upward growth of GFP-expressing E. coli in the tailpipe at an accelerated rate. The nutrient regimen that promoted colonization in our model reflects our and others' observations of items commonly disposed of in hospital sinks intravenous fluids, feeding supplements, and leftover beverages . . Transmission of bacteria between sinks via a common pipe was a key finding in this study as this highlights the concept that premise plumbing may be a more continuous system with shared microbiology than a single isolated sink.",
". Transmission of bacteria between sinks via a common pipe was a key finding in this study as this highlights the concept that premise plumbing may be a more continuous system with shared microbiology than a single isolated sink. The sink gallery used in this study provided a unique in situ advantage to investigate sink-to-sink transmission of bacteria through common drains. The two possible mechanisms for P-trap strainers becoming colonized are seeding of organisms from above and retrograde spread of organisms along common pipes in a hospital wastewater infrastructure. Here we demonstrate that it is possible for GFP-expressing E. coli to contaminate adjacent P-traps with just time and water given a standard U.S. code piping rise of 0.25 ft/ft. Sink-to-sink or retrograde transmission may explain the recurrence of pathogen colonization following intervention strategies like disinfection or replacement of plumbing .",
"Here we demonstrate that it is possible for GFP-expressing E. coli to contaminate adjacent P-traps with just time and water given a standard U.S. code piping rise of 0.25 ft/ft. Sink-to-sink or retrograde transmission may explain the recurrence of pathogen colonization following intervention strategies like disinfection or replacement of plumbing . . Sink 3 was lowest on the slope in the drain line see Fig. 4 with arguably the most opportunity for reflux and retrograde wetting. Sink 1, on the other hand, was farthest away from the source sink 5 , and its P-trap had the greatest incline in the drain line connecting the sinks, which could perhaps contribute to the reasons there was no GFP-expressing E. coli colonization detected in it after 7 days.",
"4 with arguably the most opportunity for reflux and retrograde wetting. Sink 1, on the other hand, was farthest away from the source sink 5 , and its P-trap had the greatest incline in the drain line connecting the sinks, which could perhaps contribute to the reasons there was no GFP-expressing E. coli colonization detected in it after 7 days. There has been more investigation about microbiologic dynamics of infectious viral particles such as those of severe acute respiratory syndrome SARS and Ebola viruses through premise plumbing systems . . . . However, the microbiology, sustainability, and dynamics might be very different, although the backflow and inoculation issues could have some parallels when comparing viruses to bacteria.",
". . However, the microbiology, sustainability, and dynamics might be very different, although the backflow and inoculation issues could have some parallels when comparing viruses to bacteria. As Enterobacteriaceae can either multiply or remain viable for long periods of time in biofilms coating the interior of P-traps and the connected plumbing, it may not be sustainable to target any intervention limited to a single isolated sink as a source of a particular pathogen. Data from different dispersal experiments suggest that although P-traps can act as the source or the reservoir of pathogens, the physical presence of the organism in the sink bowl or colonization of the strainer is necessary for the dispersal to occur. Colonization of strainers or drains reported in earlier studies .",
"Data from different dispersal experiments suggest that although P-traps can act as the source or the reservoir of pathogens, the physical presence of the organism in the sink bowl or colonization of the strainer is necessary for the dispersal to occur. Colonization of strainers or drains reported in earlier studies . was perhaps a result of ascending biofilm growth from the P-trap to the strainer or introduction through contaminated fluids. Many of the studies used swab samples, which likely sampled the strainer rather than P-trap water . . Once the strainer was colonized, the water from the faucet resulted in GFP-expressing E. coli dispersion in the bowl and to the surrounding surfaces of up to 30 in.",
". Once the strainer was colonized, the water from the faucet resulted in GFP-expressing E. coli dispersion in the bowl and to the surrounding surfaces of up to 30 in. The range of dispersal . . Greater dispersal near the faucet may be attributed to the specific designs of the sink bowl and faucet in this study, which determine the contact angle of water impact. This is an important finding since many sinks in hospitals are similar in design, with faucet handles representing a high-touch surface for the sink users . . It can also be concluded from the dispersion experiments that secondary and successive dispersals would likely increase the degree and the scope of dispersion.",
". It can also be concluded from the dispersion experiments that secondary and successive dispersals would likely increase the degree and the scope of dispersion. There are several limitations to this work. First the use of similar sink bowls across these sinks only examines the dispersion pattern of this particular sink design. Similarly the sink-to-sink transmission may not be applicable to all wastewater plumbing systems as the fixtures on the pipe are very close together, unlike most layouts in health care settings. However, we speculate that transmission could occur on larger systems over greater time scales, especially if heavy nutrient and contamination loads were also included.",
"Similarly the sink-to-sink transmission may not be applicable to all wastewater plumbing systems as the fixtures on the pipe are very close together, unlike most layouts in health care settings. However, we speculate that transmission could occur on larger systems over greater time scales, especially if heavy nutrient and contamination loads were also included. GFP-expressing E. coli is a laboratory surrogate, and the putative biofilms established in the short time frame of our experiments are unlikely to be as complex or stable as biofilms developed in a hospital wastewater system over many years. However, to address the monomicrobial dominance of the GFP-expressing E. coli added to the system, we kept the system open, and other environmental organisms were able to cocolonize in an attempt to mimic the hospital system. Another limitation was the need to add nutrients to the drain to ensure rapid and robust colonization. We are not clear how widespread the practice of disposing of dextrose-containing intravenous fluids or leftover beverages in the hand-wash sinks is; however, we have observed this practice, and anecdotally it appears to be relatively common in the United States.",
"Another limitation was the need to add nutrients to the drain to ensure rapid and robust colonization. We are not clear how widespread the practice of disposing of dextrose-containing intravenous fluids or leftover beverages in the hand-wash sinks is; however, we have observed this practice, and anecdotally it appears to be relatively common in the United States. We also did not completely characterize the droplet sizes, nor do we demonstrate air sampling to understand if the dispersion is only droplet or if there are also aerosols that contain GFP-expressing E. coli. This would require additional testing and is planned as future work. In summary, this work for the first time better models the mechanisms of spread of multidrug-resistant pathogens arising from the sink drain and infecting patients. Droplet dispersion from the P-trap does not happen directly.",
"In summary, this work for the first time better models the mechanisms of spread of multidrug-resistant pathogens arising from the sink drain and infecting patients. Droplet dispersion from the P-trap does not happen directly. Rather it is a multistage process: dispersal originates from the strainer and/or the bowl after growth of the biofilm up from the microbial reservoir of the P-trap. We also demonstrate sink-to-sink transmission via a common sanitary pipe. This work could have implications for patient safety, infection control, and interventions as well as the design of future hospital plumbing systems to eliminate this mode of transmission to vulnerable hospitalized patients. Sink gallery design.",
"This work could have implications for patient safety, infection control, and interventions as well as the design of future hospital plumbing systems to eliminate this mode of transmission to vulnerable hospitalized patients. Sink gallery design. A dedicated sink gallery was set up to simulate hospital hand-washing sinks. The gallery was comprised of five sink modules assembled next to each other Fig. 4 . The five hand-wash sink stations were identical in bowl designs and dimensions and were modeled from the most common intensive care unit hand-washing sink type in the acute care hospital at the University of Virginia Medical Center. Partitions made of 24-in.-high Plexiglas sheet were installed between the sinks to prevent splatter and cross contamination.",
"The five hand-wash sink stations were identical in bowl designs and dimensions and were modeled from the most common intensive care unit hand-washing sink type in the acute care hospital at the University of Virginia Medical Center. Partitions made of 24-in.-high Plexiglas sheet were installed between the sinks to prevent splatter and cross contamination. Each sink module was built with Corian integrated sink/countertops without an overflow and fitted with an 8-in. centerset 2-handle gooseneck faucet Elkay, Oak Brook, IL . The drain line Dearborn Brass-Oatey, Cleveland, OH . All of the fixtures were made of brass with chrome plating. Each of the sink P-traps was connected to a 3-in.",
"All of the fixtures were made of brass with chrome plating. Each of the sink P-traps was connected to a 3-in. common cast-iron pipe sloping into a T-joint leading into the building sanitary line located behind sink 3 Fig. 4 . Inoculation, growth, and establishment of GFP-expressing E. coli in sink P-traps. For the GFP-expressing E. coli strain ATCC 25922GFP , the green fluorescent protein GFP gene is contained on a plasmid that also contains an ampicillin resistance gene. A single isolated colony of GFP-expressing E. coli grown from a Ϫ80°C stock was inoculated into 5 ml tryptic soy broth TSB Becton, Dickinson and Company, Sparks, MD containing 100 g/ml ampicillin ATCC medium 2855 .",
"For the GFP-expressing E. coli strain ATCC 25922GFP , the green fluorescent protein GFP gene is contained on a plasmid that also contains an ampicillin resistance gene. A single isolated colony of GFP-expressing E. coli grown from a Ϫ80°C stock was inoculated into 5 ml tryptic soy broth TSB Becton, Dickinson and Company, Sparks, MD containing 100 g/ml ampicillin ATCC medium 2855 . The inoculum concentration and method varied for each experiment. For establishment of GFP-expressing E. coli in sink P-traps, new autoclaved P-traps were filled with 100 ml 0.1ϫ TSB and inoculated with ϳ10 3 CFU/ml GFPexpressing E. coli. Following inoculation, both ends of the P-traps were covered with perforated Parafilm Bemis, Inc., Oshkosh, WI and allowed to incubate at room temperature 22 Ϯ 2°C for 14 days to facilitate adherent bacterial growth. The medium in the P-trap was decanted and replaced with fresh 0.1ϫ TSB every 48 h. An aliquot of decanted medium and a swab sample from the inner surface of the P-trap were plated on tryptic soy agar Becton, Dickinson and Company, Sparks, MD plates containing 100 g/ml ampicillin TSA to monitor the growth of GFP-expressing E. coli in the P-traps.",
"Following inoculation, both ends of the P-traps were covered with perforated Parafilm Bemis, Inc., Oshkosh, WI and allowed to incubate at room temperature 22 Ϯ 2°C for 14 days to facilitate adherent bacterial growth. The medium in the P-trap was decanted and replaced with fresh 0.1ϫ TSB every 48 h. An aliquot of decanted medium and a swab sample from the inner surface of the P-trap were plated on tryptic soy agar Becton, Dickinson and Company, Sparks, MD plates containing 100 g/ml ampicillin TSA to monitor the growth of GFP-expressing E. coli in the P-traps. TSA plates were incubated overnight at 37°C, and CFU fluorescing under UV light were enumerated. All preparatory culturing of GFP-expressing E. coli took place in a separate room from the sink gallery to avoid unintentional contamination. Installation of P-traps colonized with GFP-expressing E.coli. After the 14-day incubation, P-traps were fastened into the plumbing of the sinks Fig.",
"Installation of P-traps colonized with GFP-expressing E.coli. After the 14-day incubation, P-traps were fastened into the plumbing of the sinks Fig. 5a . The remainder of the drain line was either autoclaved strainer, tailpipe, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI , maintaining at least 1 min of contact time. After the P-trap was installed, a daily regimen was followed in which 25 ml of TSB followed by 25 ml of 0.9% NaCl solution saline were added in the ratio 1:3 via the strainer Fig. 5b to mimic the potential nutrient exposure in the hospital.",
"After the P-trap was installed, a daily regimen was followed in which 25 ml of TSB followed by 25 ml of 0.9% NaCl solution saline were added in the ratio 1:3 via the strainer Fig. 5b to mimic the potential nutrient exposure in the hospital. Sampling and enumeration of GFP-expressing E. coli. To monitor the growth of GFP-expressing E. coli in the plumbing, sampling ports were drilled along the length of the tailpiece between the P-trap and the strainer and the trap arm between the P-trap and the common line . These holes were fitted with size 00 silicone stoppers Cole-Parmer, Vernon Hills, IL Fig. 5a .",
"These holes were fitted with size 00 silicone stoppers Cole-Parmer, Vernon Hills, IL Fig. 5a . Sterile cotton swabs Covidien, Mansfield, MA presoaked in saline were inserted through these sampling ports, and samples were collected by turning the swab in a circular motion on the inner surface ϳ20 cm 2 of the tailpipes. Sample swabs were pulse-vortexed in 3 ml saline, and serial dilutions were plated on TSA. The strainer, faucet aerator, and bowl surface were sampled with presoaked swabs and processed as described earlier. Sink-to-sink transmission of bacteria. To investigate sink-to-sink transmission of bacteria, a distal sink sink 5 Fig.",
"Sink-to-sink transmission of bacteria. To investigate sink-to-sink transmission of bacteria, a distal sink sink 5 Fig. 4 was fitted with a P-trap inoculated with GFP-expressing E. coli. The effects of different inoculum concentrations of GFP-expressing E. coli-10 3 , 10 6 , and Ͼ10 10 CFU/ml colonized for 14 days -were investigated. Identification to the species level of fluorescent and nonfluorescent colonies identified from mixed pipe cultures was performed using a matrix-assisted laser desorption-ionization MALDI -time of flight MALDI-TOF mass spectrometer Vitek-MS; bioMérieux, Durham, NC . The wastewater paths of sinks 1 to 4 were either autoclaved strainer, tailpipe, P-traps, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI .",
"Identification to the species level of fluorescent and nonfluorescent colonies identified from mixed pipe cultures was performed using a matrix-assisted laser desorption-ionization MALDI -time of flight MALDI-TOF mass spectrometer Vitek-MS; bioMérieux, Durham, NC . The wastewater paths of sinks 1 to 4 were either autoclaved strainer, tailpipe, P-traps, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI . Faucets on each of the five sinks were turned on simultaneously for 1 min, supplying water at a flow rate of 8 liters/min, once every 24 h for 7 days. No additional feed to any of the sinks was added during this period of 7 days. On days 0 and 7, P-traps on each of the five sinks were unfastened, and swab samples from the P-trap were collected and processed as described earlier. Dispersion measured using fluorescent microspheres.",
"On days 0 and 7, P-traps on each of the five sinks were unfastened, and swab samples from the P-trap were collected and processed as described earlier. Dispersion measured using fluorescent microspheres. Fluoresbrite YO carboxylate microspheres Polysciences, Inc. which had a 1-m diameter and maximum excitation and emission of 529 nm and 546 nm, respectively, were used as a tracer in the preliminary experiments to understand droplet dispersion from the hand-wash sinks. To test whether microspheres could be dispersed from below the sink strainer, a 1-ml suspension of microspheres ϳ10 10 particles was injected through a strainer attached to a Hert 4.5-in. offset drain tailpiece typically used for wheelchair-accessible sinks American Standard, model 7723018.002 Fig. 5c .",
"offset drain tailpiece typically used for wheelchair-accessible sinks American Standard, model 7723018.002 Fig. 5c . The vertical distance between the strainer and microsphere suspension injected into the tailpipe was ϳ4 in. Counter space around the sink bowl was thoroughly wiped with alcohol wipes Covidien Webcol 6818; Kendall , and polyester sheets precut to appropriate shapes were placed on the counter to cover the entire sink counter and labeled according to position Fig. 6a . The faucet was turned on for 5 min at a water flow rate of 1.8 to 3.0 liters/min.",
"6a . The faucet was turned on for 5 min at a water flow rate of 1.8 to 3.0 liters/min. Polyester sheets were harvested and immediately analyzed using a ChemiDoc MP system Bio-Rad Laboratories, Inc. with an exposure time of 5 s. Fluorescent microspheres were enumerated from the digital micrographs using the Image Lab Software Bio-Rad Laboratories, Inc. . To test whether microspheres could be dispersed from the surface of the sink bowl, the sink bowl was evenly coated with a 20-ml microsphere suspension ϳ10 10 particles/ml using a disposable swab Sage Products, Inc., Cary, IL , and the dispersion experiment was repeated following the protocol described above. To ascertain there was no nonspecific background fluorescence in the sink and/or the water from the faucet, a control using the same protocol but without the fluorescent microspheres was performed before each experiment. Dispersion measured using GFP-expressing E.coli.",
"To ascertain there was no nonspecific background fluorescence in the sink and/or the water from the faucet, a control using the same protocol but without the fluorescent microspheres was performed before each experiment. Dispersion measured using GFP-expressing E.coli. Dispersion using GFP-expressing E. coli was investigated in three experiments. To test whether live organisms in the P-trap could be dispersed by running water, ϳ10 10 CFU/ml GFP-expressing E. coli in saline was added to an autoclaved P-trap and fitted into the drain line that was preautoclaved strainer, tailpipe, and trap arms . Similarly, to test whether live organisms could be dispersed from the tailpieces of wheelchair-accessible sinks, a suspension of ϳ10 10 CFU/ml GFP-expressing E. coli was added with a syringe through the strainer into the Hert 4.5-ft offset drain tailpiece Fig. 5c .",
"Similarly, to test whether live organisms could be dispersed from the tailpieces of wheelchair-accessible sinks, a suspension of ϳ10 10 CFU/ml GFP-expressing E. coli was added with a syringe through the strainer into the Hert 4.5-ft offset drain tailpiece Fig. 5c . Just as in the microsphere dispersion experiment, the vertical distance between the strainer and GFP-expressing E. coli suspension injected into the tailpipe was ϳ4 in. We next tested whether live organisms from the surface of the sink bowl could be dispersed by running water. The sink bowl surface was evenly coated with an approximately 20-ml suspension of 10 10 CFU/ml GFP-expressing E. coli. Finally, to mimic all of these conditions, a P-trap colonized with GFP-expressing E. coli for 14 days was installed, and a nutrient regimen Fig.",
"The sink bowl surface was evenly coated with an approximately 20-ml suspension of 10 10 CFU/ml GFP-expressing E. coli. Finally, to mimic all of these conditions, a P-trap colonized with GFP-expressing E. coli for 14 days was installed, and a nutrient regimen Fig. 5b was followed for 14 days to intentionally promote the GFP-expressing E. coli colonization in the attached tailpipe and strainer. On day 15, the dispersion experiment was performed. Before each of the GFP-expressing E. coli dispersion experiments, the counter space was thoroughly disinfected with Caviwipes-1. TSA plates were then positioned on the sink counter surrounding the bowl and an extension platform Fig. 6b .",
"TSA plates were then positioned on the sink counter surrounding the bowl and an extension platform Fig. 6b . Additional plates were attached to the sink bowl, faucets, Plexiglas partitions, and faucet handles using adhesive tape. TSA plates were also placed 3 m away from the sink as negative controls. The faucet was turned on for 5 min with a water flow rate of 1.8 to 3.0 liters/min. Lids of the TSA plates were removed only during faucet operation. Swab samples from the faucet aerators before and after operation were collected and plated on TSA.",
"Lids of the TSA plates were removed only during faucet operation. Swab samples from the faucet aerators before and after operation were collected and plated on TSA. Prior to each dispersion experiment, 50 ml water from the faucet was also collected, and aliquots were plated to assess for the presence of GFP-expressing E. coli in source water and ensure cross contamination of GFP-expressing E. coli had not occurred. A control dispersion experiment was also performed using the same protocol prior to GFP-expressing E. coli inoculation in each case. Dispersion per defined area CFU per square centimeter was deduced by dividing the CFU counts in the TSA plate by the surface area of the TSA plate."
] | 2,585 | 545 |
What types of acute respiratory infections can be screened and diagnosed with multiplex PCR?
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influenza and parainfluenza viruses, RSV, adenovirus, rhinovirus, human metapneumovirus, enterovirus, bocavirus and coronavirus
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[
"OBJECTIVE: To determine whether rapid polymerase chain reaction PCR testing for influenza and respiratory syncytial viruses RSV in emergency departments EDs is associated with better patient and laboratory outcomes than standard multiplex PCR testing. DESIGN, SETTING: A before‐and‐after study in four metropolitan EDs in New South Wales. PARTICIPANTS: 1491 consecutive patients tested by standard multiplex PCR during July–December 2016, and 2250 tested by rapid PCR during July–December 2017. MAIN OUTCOME MEASURES: Hospital admissions; ED length of stay LOS ; test turnaround time; patient receiving test result before leaving the ED; ordering of other laboratory tests. RESULTS: Compared with those tested by standard PCR, fewer patients tested by rapid PCR were admitted to hospital 73.3% v 77.7%; P < 0.001 and more received their test results before leaving the ED 67.4% v 1.3%; P < 0.001 ; the median test turnaround time was also shorter 2.4 h IQR, 1.6–3.9 h v 26.7 h IQR, 21.2–37.8 h . The proportion of patients admitted to hospital was also lower in the rapid PCR group for both children under 18 50.6% v 66.6%; P < 0.001 and patients over 60 years of age 84.3% v 91.8%; P < 0.001 .",
"RESULTS: Compared with those tested by standard PCR, fewer patients tested by rapid PCR were admitted to hospital 73.3% v 77.7%; P < 0.001 and more received their test results before leaving the ED 67.4% v 1.3%; P < 0.001 ; the median test turnaround time was also shorter 2.4 h IQR, 1.6–3.9 h v 26.7 h IQR, 21.2–37.8 h . The proportion of patients admitted to hospital was also lower in the rapid PCR group for both children under 18 50.6% v 66.6%; P < 0.001 and patients over 60 years of age 84.3% v 91.8%; P < 0.001 . Significantly fewer blood culture, blood gas, sputum culture, and respiratory bacterial and viral serology tests were ordered for patients tested by rapid PCR. ED LOS was similar for the rapid 7.4 h; IQR, 5.0–12.9 h and standard PCR groups 6.5 h; IQR, 4.2–11.9 h; P = 0.27 . CONCLUSION: Rapid PCR testing of ED patients for influenza virus and RSV was associated with better outcomes on a range of indicators, suggesting benefits for patients and the health care system. A formal cost–benefit analysis should be undertaken.",
"CONCLUSION: Rapid PCR testing of ED patients for influenza virus and RSV was associated with better outcomes on a range of indicators, suggesting benefits for patients and the health care system. A formal cost–benefit analysis should be undertaken. Text: The health and economic burdens associated with acute respiratory infections by influenza and respiratory syncytial viruses RSV are significant in Australia and overseas. 1-3 Polymerase chain reaction PCR testing is effective for confirming respiratory viral infections. 4 Multiplex PCR can detect numerous respiratory viruses, including influenza and parainfluenza viruses, RSV, adenovirus, rhinovirus, human metapneumovirus, enterovirus, bocavirus and coronavirus with very high sensitivity and specificity. 5 Although the results of standard multiplex PCR are accurate and comprehensive, it has traditionally been performed in a central laboratory with a lengthy turnaround time, which may be inconvenient in settings where test results are urgently required, including emergency departments EDs .",
"4 Multiplex PCR can detect numerous respiratory viruses, including influenza and parainfluenza viruses, RSV, adenovirus, rhinovirus, human metapneumovirus, enterovirus, bocavirus and coronavirus with very high sensitivity and specificity. 5 Although the results of standard multiplex PCR are accurate and comprehensive, it has traditionally been performed in a central laboratory with a lengthy turnaround time, which may be inconvenient in settings where test results are urgently required, including emergency departments EDs . Rapid, easy-to-use PCR-based respiratory virus diagnostic tests have been introduced in recent years; 6,7 the GeneXpert system Cepheid , for instance, was introduced in New South Wales in July 2017. Rapid PCR tests were expected to facilitate timely and appropriate initiation of treatment, improve outbreak prevention and infection control measures, and expedite the assessment of patients in EDs. In this study, we analysed routinely collected data to determine whether rapid PCR testing for influenza and RSV infections in EDs is associated with improved patient and laboratory outcomes. We compared data for patients tested for influenza A/B viruses and RSV immediately after rapid PCR diagnosis was introduced July-December 2017 with data for patients tested with a standard multiplex PCR system during July-December 2016.",
"In this study, we analysed routinely collected data to determine whether rapid PCR testing for influenza and RSV infections in EDs is associated with improved patient and laboratory outcomes. We compared data for patients tested for influenza A/B viruses and RSV immediately after rapid PCR diagnosis was introduced July-December 2017 with data for patients tested with a standard multiplex PCR system during July-December 2016. We undertook a before-and-after study in four metropolitan public hospital EDs in Sydney, NSW: three general hospitals EDs A, B and C; 76 228, 54 443 and 50 025 annual ED presentations respectively and one children's hospital ED D; 36 700 annual ED presentations; all data for January-December 2016 8 . The four hospitals were served by a single pathology laboratory provider. We analysed data for all patients tested for influenza virus or RSV. During July-December 2016, patients were tested with the standard PCR system, a central laboratory-based multiplex PCR test for sixteen respiratory viruses including RSV and influenza viruses A and B , available as a referral test at the central laboratory in Hospital B.",
"We analysed data for all patients tested for influenza virus or RSV. During July-December 2016, patients were tested with the standard PCR system, a central laboratory-based multiplex PCR test for sixteen respiratory viruses including RSV and influenza viruses A and B , available as a referral test at the central laboratory in Hospital B. During July-December 2017, patients were tested with the rapid PCR system, a hospital laboratory-based test specific for RSV and influenza viruses A and B. Hospitals A, B and D have onsite laboratories that perform rapid PCR testing; Hospital C sends samples to the nearby Hospital A. All tests were conducted in virology laboratories by trained staff, and test results were entered into laboratory information system datasets. We obtained relevant patient characteristics and The proportion of patients admitted to hospital was also lower in the rapid PCR group for both children under 18 50.6% v 66.6%; P < 0.001 and patients over 60 years of age 84.3% v 91.8%; P < 0.001 .",
"All tests were conducted in virology laboratories by trained staff, and test results were entered into laboratory information system datasets. We obtained relevant patient characteristics and The proportion of patients admitted to hospital was also lower in the rapid PCR group for both children under 18 50.6% v 66.6%; P < 0.001 and patients over 60 years of age 84.3% v 91.8%; P < 0.001 . Significantly fewer blood culture, blood gas, sputum culture, and respiratory bacterial and viral serology tests were ordered for patients tested by rapid PCR. ED LOS was similar for the rapid 7.4 h; IQR, 5.0-12.9 h and standard PCR groups 6.5 h; IQR, 4.2-11.9 h; P = 0.27 . Conclusion: Rapid PCR testing of ED patients for influenza virus and RSV was associated with better outcomes on a range of indicators, suggesting benefits for patients and the health care system. A formal cost-benefit analysis should be undertaken.",
"Conclusion: Rapid PCR testing of ED patients for influenza virus and RSV was associated with better outcomes on a range of indicators, suggesting benefits for patients and the health care system. A formal cost-benefit analysis should be undertaken. The known: Rapid polymerase chain reaction PCR testing for influenza and respiratory syncytial viruses RSV was introduced in New South Wales in July 2017. Its impact on outcomes for emergency department ED patients has not been investigated. The new: Compared with standard PCR testing, rapid PCR was associated with significantly fewer hospital admissions, more rapid test turnaround, more patients receiving test results before leaving the ED, and reduced numbers of some other common microbiology tests. It did not significantly affect ED length of stay.",
"The new: Compared with standard PCR testing, rapid PCR was associated with significantly fewer hospital admissions, more rapid test turnaround, more patients receiving test results before leaving the ED, and reduced numbers of some other common microbiology tests. It did not significantly affect ED length of stay. The implications: Rapid PCR testing of ED patients for major respiratory viruses can benefit patients and reduce resource use. MJA 210 . ▪ 15 April 2019 317 laboratory data by linking the ED and laboratory information system datasets. Detailed information about the datasets and the linkage process have been described previously.",
"▪ 15 April 2019 317 laboratory data by linking the ED and laboratory information system datasets. Detailed information about the datasets and the linkage process have been described previously. 9 The primary outcomes were admission to hospital, ED length of stay LOS , test turnaround time, and the patient receiving their test result before leaving the ED. ED LOS was defined as the time from arrival in the ED to discharge or admission to hospital. Turnaround was defined as the time from when the sample was received by the laboratory to when the test result was available in hospital electronic records. As secondary outcomes, we compared ordering of typical biochemistry and haematology tests full blood count; electrolyte, urea, creatinine levels; liver function test; blood gas analysis; C-reactive protein level and microbiology tests blood culture; urine microscopy, culture and sensitivity analysis; sputum culture; respiratory bacteria and virus serology during the two study periods.",
"Turnaround was defined as the time from when the sample was received by the laboratory to when the test result was available in hospital electronic records. As secondary outcomes, we compared ordering of typical biochemistry and haematology tests full blood count; electrolyte, urea, creatinine levels; liver function test; blood gas analysis; C-reactive protein level and microbiology tests blood culture; urine microscopy, culture and sensitivity analysis; sputum culture; respiratory bacteria and virus serology during the two study periods. Analyses were conducted in Stata 15 StataCorp . Descriptive statistics are reported medians with interquartile ranges IQRs , means with standard deviations SDs , numbers with proportions . Baseline characteristics were compared in χ 2 tests categorical outcomes or Wilcoxon rank-sum tests continuous outcomes . Logistic regression analyses of binary outcomes eg, hospital admission: yes/no and quantile regression analyses of continuous outcomes eg, ED LOS were undertaken.",
"Baseline characteristics were compared in χ 2 tests categorical outcomes or Wilcoxon rank-sum tests continuous outcomes . Logistic regression analyses of binary outcomes eg, hospital admission: yes/no and quantile regression analyses of continuous outcomes eg, ED LOS were undertaken. All analyses were adjusted for baseline characteristics. Sensitivity analyses limited to data for children under 18 years of age or older patients over 60 years of age were conducted. P < 0.05 2-tailed was deemed statistically significant. Ethics approval for the investigation was granted by the Human Research Ethics Committee of the South Eastern Sydney Local Health District reference, HREC/16/POWH/412 .",
"P < 0.05 2-tailed was deemed statistically significant. Ethics approval for the investigation was granted by the Human Research Ethics Committee of the South Eastern Sydney Local Health District reference, HREC/16/POWH/412 . We analysed data for 3741 patients presenting to the four EDs during two periods: 1491 consecutive patients during July-December 2016 standard PCR and 2250 during July-December 2017 rapid PCR . Baseline characteristics for the two groups were similar in terms of sex, triage category, and arrival day of the week, but differed significantly for age, arrival time, and mode of arrival Box 1 The overall numbers of tests per patient were similar in the standard PCR mean, 7.2 tests; SD, 3.8 and rapid PCR groups mean, 7.1 tests; SD, 3.4 . The mean number of microbiology tests per patient was significantly lower for the rapid PCR group 1.5 tests; SD, 1.8 than for the standard PCR group 2.0 tests; SD, 2.1; P < 0.001 after controlling for baseline characteristics . The 16 265 biochemistry/haematology and microbiology tests comprised 71.1% of the 22 876 other tests that is, not including PCR virus testing ordered for patients in the study.",
"The mean number of microbiology tests per patient was significantly lower for the rapid PCR group 1.5 tests; SD, 1.8 than for the standard PCR group 2.0 tests; SD, 2.1; P < 0.001 after controlling for baseline characteristics . The 16 265 biochemistry/haematology and microbiology tests comprised 71.1% of the 22 876 other tests that is, not including PCR virus testing ordered for patients in the study. After adjusting for baseline characteristics, the proportions of patients for whom full blood count, electrolyte/urea/creatinine levels, liver function, or C-reactive protein were assessed were similar, as were the proportions for urine microscopy, culture and sensitivity tests. Significantly fewer blood culture, blood gas, sputum culture, and respiratory bacterial and viral serology tests were ordered for patients in the rapid PCR group Box 4 . Information, figure 1 . ED LOS was similar for the standard PCR and rapid PCR groups in both age-based subgroups Supporting Information, figure 2A .",
"Information, figure 1 . ED LOS was similar for the standard PCR and rapid PCR groups in both age-based subgroups Supporting Information, figure 2A . The differences in test turnaround time identified in the main analysis were also evident for each age-based subgroup Supporting Information, figure 2B . In this before-and-after study, we found that rapid PCR testing of ED patients for major respiratory viruses was associated with significantly fewer admissions to hospital, more rapid delivery of test results, more patients receiving their test results before leaving the ED, and less frequent ordering of some common microbiology tests. Other studies have also reported that hospital admission numbers were significantly lower when rapid influenza virus testing was used in EDs. An analysis of outcomes for more than 300 adults at a tertiary care centre in New York found that early diagnosis of respiratory infections was associated with significantly fewer hospitalisations of influenza-positive patients.",
"Other studies have also reported that hospital admission numbers were significantly lower when rapid influenza virus testing was used in EDs. An analysis of outcomes for more than 300 adults at a tertiary care centre in New York found that early diagnosis of respiratory infections was associated with significantly fewer hospitalisations of influenza-positive patients. 7 In a small Irish study 73 patients , the hospital admission rate for obstetric patients declined from 88% to 45% after on-site rapid influenza PCR testing was introduced. 10 The differences in clinical setting and patient group may explain the smaller decline in our study from 78% to 67% . Non-PCR-based rapid diagnostic tests for respiratory viruses have also been associated with lower hospital admission rates. 11, 12 The main reason for fewer hospital admissions of patients tested by rapid PCR may be that the earlier availability of results enables clinicians to quickly diagnose or exclude respiratory infections and to make timely and informed decisions about whether to discharge the patient or admit them to hospital.",
"Non-PCR-based rapid diagnostic tests for respiratory viruses have also been associated with lower hospital admission rates. 11, 12 The main reason for fewer hospital admissions of patients tested by rapid PCR may be that the earlier availability of results enables clinicians to quickly diagnose or exclude respiratory infections and to make timely and informed decisions about whether to discharge the patient or admit them to hospital. When standard 2 Primary outcomes for 3741 patients at four metropolitan emergency departments EDs tested for influenza and respiratory syncytial viruses by standard or rapid polymerase chain reaction PCR After adjusting for baseline characteristics Box 1 : * P = 0.012; ** P < 0.001. ◆ MJA 210 . ▪ 15 April 2019 PCR was used, in contrast, our findings suggest that these decisions were made before the test results were available. The possible benefits of not admitting patients to hospital, beyond those for individual patient management, include better infection control and outbreak prevention, as well as reduced demands on hospital resources.",
"▪ 15 April 2019 PCR was used, in contrast, our findings suggest that these decisions were made before the test results were available. The possible benefits of not admitting patients to hospital, beyond those for individual patient management, include better infection control and outbreak prevention, as well as reduced demands on hospital resources. 13, 14 The impact of rapid PCR testing on outbreak prevention and infection control measures should be evaluated. Rapid influenza virus testing may also have practical implications for hospital bed management. 10, 15 ED LOS was similar in our study before and after the introduction of rapid PCR methods. This finding was not unexpected, as test turnaround time is not the only rate-limiting factor for decision making in EDs.",
"10, 15 ED LOS was similar in our study before and after the introduction of rapid PCR methods. This finding was not unexpected, as test turnaround time is not the only rate-limiting factor for decision making in EDs. 16 Before rapid PCR methods were introduced, the long turnaround time of multiplex PCR did not necessarily extend a patient's stay in the ED, as they were usually admitted to hospital or discharged home before the results were available. Consequently, more rapid delivery of test results alone would not reduce ED LOS. Reports on the effect of rapid influenza virus testing and LOS have been conflicting. While evidence for an association between rapid testing and shorter overall inpatient LOS has been reported, 6,11 findings regarding ED LOS are inconsistent.",
"Reports on the effect of rapid influenza virus testing and LOS have been conflicting. While evidence for an association between rapid testing and shorter overall inpatient LOS has been reported, 6,11 findings regarding ED LOS are inconsistent. 7, 17, 18 For example, a Cochrane review based on three randomised controlled trials did not find a statistically significant association of rapid viral diagnosis with lower mean ED LOS. 18 In a study in children, ED LOS was actually 26 minutes longer with rapid PCR; inpatient LOS did not differ between the two groups, but was significantly shorter when the analysis was limited to patients with positive test results. 6 We found that ordering of some other microbiology tests, including blood culture, sputum culture, and respiratory bacterial and virus serology, was significantly less frequent for patients tested by rapid PCR. The effect of PCR-based rapid testing on the ordering of other laboratory tests has not previously been reported, although some studies of antigen-based pointof-care testing have examined this outcome.",
"6 We found that ordering of some other microbiology tests, including blood culture, sputum culture, and respiratory bacterial and virus serology, was significantly less frequent for patients tested by rapid PCR. The effect of PCR-based rapid testing on the ordering of other laboratory tests has not previously been reported, although some studies of antigen-based pointof-care testing have examined this outcome. 12 Consistent with our finding, several investigators have reported fewer blood culture tests 19, 20 and basic biochemistry and haematology tests, including full blood count, 20,21 C-reactive protein testing, 21 and urinalysis, 20,21 when point-of-care testing was used. The higher rate of positive results for patients tested by rapid PCR than for those tested by standard PCR may reflect a higher prevalence of influenza during 2017 than in 2016. The rapid PCR system in our study accurately detects influenza viruses A/B and RSV but, unlike the standard multiplex PCR, cannot detect other clinically relevant respiratory viruses, such as rhinovirus, coronavirus, human metapneumovirus, parainfluenza virus, adenovirus, enterovirus, and bocavirus. If infection with other respiratory viruses is suspected, patients may therefore need further investigations.",
"The rapid PCR system in our study accurately detects influenza viruses A/B and RSV but, unlike the standard multiplex PCR, cannot detect other clinically relevant respiratory viruses, such as rhinovirus, coronavirus, human metapneumovirus, parainfluenza virus, adenovirus, enterovirus, and bocavirus. If infection with other respiratory viruses is suspected, patients may therefore need further investigations. Standard multiplex PCR can provide broader information, as it can detect multiple respiratory viruses in a single run, although the long turnaround time restricts its suitability for urgent clinical decision making. Improving the turnaround time of multiplex PCR analysis may achieve better outcomes. The strengths of our study include its relatively large sample size; further, unlike many similar investigations, ours was a multicentre study in four hospital EDs, enhancing the generalisability of our findings. However, our analyses were not adjusted for comorbid conditions, as this information was not available.",
"The strengths of our study include its relatively large sample size; further, unlike many similar investigations, ours was a multicentre study in four hospital EDs, enhancing the generalisability of our findings. However, our analyses were not adjusted for comorbid conditions, as this information was not available. Because our study was an uncontrolled before-and-after study, our results cannot be interpreted as indicating causal relationships. As with all non-randomised trials, we could not fully account for all potential confounding variables, nor for temporal trends and other unmeasured factors, such as changes in clinician testing practices or differences in the prevalence and severity of disease during the two study periods. 22 For example, a shortage of inpatient beds caused by a high prevalence of influenza could influence decisions in a busy ED about admitting patients to hospital. However, we attempted to reduce seasonal effects by selecting similar time frames for the two study periods, to reduce selection bias by including all ED patients tested for influenza virus and RSV, and to control for differences in baseline patient characteristics by applying multivariate modelling.",
"22 For example, a shortage of inpatient beds caused by a high prevalence of influenza could influence decisions in a busy ED about admitting patients to hospital. However, we attempted to reduce seasonal effects by selecting similar time frames for the two study periods, to reduce selection bias by including all ED patients tested for influenza virus and RSV, and to control for differences in baseline patient characteristics by applying multivariate modelling. As medications data were not available to us, we were unable to assess the impact of rapid PCR testing on antibiotic and antiviral drug use. Similarly, the cost-benefit balance of rapid testing was not evaluated because relevant data were not available. The cost per patient of rapid PCR testing is generally higher than for central laboratory testing, but our findings suggest potential savings through lower numbers of hospital admissions and reduced resource use. This question could be evaluated in a further study.",
"The cost per patient of rapid PCR testing is generally higher than for central laboratory testing, but our findings suggest potential savings through lower numbers of hospital admissions and reduced resource use. This question could be evaluated in a further study. Rapid PCR testing for influenza virus and RSV infections in patients attending EDs was associated with significant improvements in a range of patient and laboratory outcomes, suggesting potential benefits for both the patients and the health care system. A cost-benefit analysis could examine the impact of rapid PCR testing on bed management and antimicrobial drug prescribing."
] | 1,556 | 327 |
What is the role of antibodies during infection?
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Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections.
|
[
"Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.",
"These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .",
"Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .",
"Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .",
"Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.",
"antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .",
". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .",
". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.",
"Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.",
"Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.",
"The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.",
"In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .",
"Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .",
"In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .",
"Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.",
". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.",
"Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .",
"Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .",
"Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.",
". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.",
"Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.",
". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .",
"The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.",
"Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .",
". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .",
"Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.",
"choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .",
"Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .",
"Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.",
". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .",
"Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.",
"Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .",
"Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .",
"On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .",
". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript."
] | 1,569 | 465 |
How can antibodies also create health problems?
|
Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection.
|
[
"Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.",
"These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .",
"Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .",
"Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .",
"Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.",
"antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .",
". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .",
". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.",
"Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.",
"Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.",
"The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.",
"In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .",
"Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .",
"In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .",
"Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.",
". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.",
"Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .",
"Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .",
"Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.",
". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.",
"Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.",
". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .",
"The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.",
"Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .",
". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .",
"Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.",
"choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .",
"Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .",
"Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.",
". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .",
"Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.",
"Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .",
"Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .",
"On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .",
". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript."
] | 1,569 | 467 |
Which technology invention produced antibodies that are clones of a unique parent cell?
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in the 1970s with the development of hybridoma technology to produce monoclonal antibodies
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[
"Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.",
"These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .",
"Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .",
"Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .",
"Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.",
"antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .",
". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .",
". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.",
"Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.",
"Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.",
"The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.",
"In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .",
"Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .",
"In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .",
"Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.",
". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.",
"Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .",
"Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .",
"Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.",
". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.",
"Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.",
". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .",
"The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.",
"Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .",
". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .",
"Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.",
"choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .",
"Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .",
"Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.",
". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .",
"Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.",
"Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .",
"Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .",
"On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .",
". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript."
] | 1,569 | 474 |
What mechanism is responsible for the creation of diversified repertoire for antibodies?
|
somatic rearrangement during B cell differentiation was responsible for antibody diversification
|
[
"Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.",
"These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .",
"Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .",
"Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .",
"Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.",
"antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .",
". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .",
". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.",
"Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.",
"Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.",
"The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.",
"In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .",
"Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .",
"In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .",
"Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.",
". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.",
"Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .",
"Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .",
"Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.",
". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.",
"Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.",
". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .",
"The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.",
"Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .",
". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .",
"Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.",
"choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .",
"Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .",
"Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.",
". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .",
"Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.",
"Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .",
"Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .",
"On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .",
". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript."
] | 1,569 | 476 |
What developments have been made possible by the study of B-cell repertoire?
|
(1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease.
|
[
"Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.",
"These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .",
"Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .",
"Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .",
"Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.",
"antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .",
". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .",
". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.",
"Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.",
"Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.",
"The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.",
"In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .",
"Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .",
"In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .",
"Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.",
". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.",
"Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .",
"Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .",
"Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.",
". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.",
"Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.",
". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .",
"The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.",
"Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .",
". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .",
"Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.",
"choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .",
"Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .",
"Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.",
". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .",
"Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.",
"Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .",
"Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .",
"On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .",
". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript."
] | 1,569 | 478 |
What motivates the study of the rare B-cells that produce Broadly Neutralizing Antibodies (bnAb)?
|
discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies
|
[
"Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.",
"These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .",
"Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .",
"Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .",
"Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.",
"antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .",
". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .",
". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.",
"Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.",
"Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.",
"The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.",
"In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .",
"Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .",
"In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .",
"Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.",
". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.",
"Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .",
"Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .",
"Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.",
". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.",
"Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.",
". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .",
"The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.",
"Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .",
". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .",
"Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.",
"choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .",
"Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .",
"Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.",
". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .",
"Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.",
"Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .",
"Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .",
"On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .",
". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript."
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How has the study of B-cells helped the treatment for Respiratory syncytial virus (RSV)?
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For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates
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"Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.",
"These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .",
"Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .",
"Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .",
"Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.",
"antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .",
". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .",
". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.",
"Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.",
"Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.",
"The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.",
"In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .",
"Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .",
"In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .",
"Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.",
". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.",
"Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .",
"Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .",
"Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.",
". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.",
"Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.",
". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .",
"The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.",
"Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .",
". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .",
"Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.",
"choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .",
"Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .",
"Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.",
". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .",
"Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.",
"Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .",
"Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .",
"On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .",
". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript."
] | 1,569 | 489 |
How are the studies on B-cells helping the development of a universal influenza vaccine?
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Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (
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[
"Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.",
"These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .",
"Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .",
"Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .",
"Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.",
"antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .",
". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .",
". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.",
"Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.",
"Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.",
"The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.",
"In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .",
"Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .",
"In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .",
"Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.",
". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.",
"Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .",
"Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .",
"Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.",
". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.",
"Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.",
". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .",
"The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.",
"Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .",
". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .",
"Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.",
"choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .",
"Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .",
"Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.",
". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .",
"Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.",
"Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .",
"Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .",
"On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .",
". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript."
] | 1,569 | 490 |
What role B-cell play in malaria infection and prevention?
|
Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection
|
[
"Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.",
"These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .",
"Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .",
"Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .",
"Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.",
"antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .",
". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .",
". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.",
"Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.",
"Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.",
"The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.",
"In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .",
"Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .",
"In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .",
"Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.",
". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.",
"Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .",
"Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .",
"Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.",
". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.",
"Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.",
". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .",
"The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.",
"Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .",
". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .",
"Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.",
"choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .",
"Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .",
"Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.",
". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .",
"Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.",
"Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .",
"Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .",
"On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .",
". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript."
] | 1,569 | 494 |
How can the study of B-cells help in the prevention and treatment of autoimmune diseases?
|
The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.
|
[
"Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.",
"These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .",
"Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .",
"Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .",
"Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.",
"antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .",
". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .",
". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.",
"Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.",
"Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.",
"The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.",
"In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .",
"Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .",
"In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .",
"Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.",
". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.",
"Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .",
"Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .",
"Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.",
". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.",
"Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.",
". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .",
"The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.",
"Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .",
". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .",
"Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.",
"choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .",
"Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .",
"Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.",
". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .",
"Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.",
"Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .",
"Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .",
"On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .",
". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript."
] | 1,569 | 497 |
Why are cotton rats considered a strong animal model for biomedical research?
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its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents
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[
"Cotton rats are an important animal model to study infectious diseases. They have demonstrated higher susceptibility to a wider variety of human pathogens than other rodents and are also the animal model of choice for pre-clinical evaluations of some vaccine candidates. However, the genome of cotton rats remains to be fully sequenced, with much fewer genes cloned and characterised compared to other rodent species. Here we report the cloning and characterization of CD40 ligand, whose human and murine counterparts are known to be expressed on a range of cell types including activated T cells and B cells, dendritic cells, granulocytes, macrophages and platelets and exerts a broad array of immune responses. The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame ORF of 783bp coding for a 260 amino acid protein. The recombinant cotton rat CD40L protein was recognized by an antibody against mouse CD40L.",
"The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame ORF of 783bp coding for a 260 amino acid protein. The recombinant cotton rat CD40L protein was recognized by an antibody against mouse CD40L. Moreover, it demonstrated functional activities on immature bone marrow dendritic cells by upregulating surface maturation markers CD40, CD54, CD80, and CD86 , and increasing IL-6 gene and protein expression. The availability of CD40L gene identity could greatly facilitate mechanistic research on pathogen-induced-immunopathogenesis and vaccine-elicited immune responses. Text: The cotton rat Sigmodon hispidus was first used in polio research in the 1930s , and throughout the last century, it has proven to be an excellent model for biomedical research . Historically in biomedical research, the mouse has been exploited as the default animal model.",
"Text: The cotton rat Sigmodon hispidus was first used in polio research in the 1930s , and throughout the last century, it has proven to be an excellent model for biomedical research . Historically in biomedical research, the mouse has been exploited as the default animal model. This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus . This model has been valuable for adenovirus-based gene replacement therapy research , and was also proven to be indispensable in pre-clinical evaluation of the prophylactic antibodies RespiGam 1 , and Synagis 1 .",
"On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus . This model has been valuable for adenovirus-based gene replacement therapy research , and was also proven to be indispensable in pre-clinical evaluation of the prophylactic antibodies RespiGam 1 , and Synagis 1 . Indeed, the cotton rat model was found to be valuable in terms of its biological and immunological relevance, it was deemed unnecessary to test the adenovirus-based gene therapy and the Synagis 1 prophylactic treatment against RSV disease in non-human primate prior to the human trials . A number of methods and reagents have been developed for the analysis of immune responses in cotton rats over the last decade. Up to date, more than 200 genes encoding cytokines, chemokines, cell surface markers and regulatory molecules have been cloned, with various related research reagents being commercially available. As a result, the use of cotton rats in pathogenesis studies addressing mechanistic questions has significantly increased.",
"Up to date, more than 200 genes encoding cytokines, chemokines, cell surface markers and regulatory molecules have been cloned, with various related research reagents being commercially available. As a result, the use of cotton rats in pathogenesis studies addressing mechanistic questions has significantly increased. Nevertheless, the gene encoding CD154 and CD40 ligand CD40L , remains elusive. CD40L plays a critical role in orchestrating immune responses against pathogens. Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands .",
"Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets . It has been shown that upon interacting with its receptor, CD40, CD40L induces profound effects on T cells, DCs, B cells, endothelial cells, as well as many cells of the hematopoietic and non-hematopoietic systems. Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin Ig isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy.",
"Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin Ig isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy. It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes . Here we report the successful cloning of the gene encoding cotton rat CD40L crCD40L ; we also expressed and purified the CD40L produced in mammalian cells. Further characterisation of the recombinant cotton rat CD40L revealed its functional activities in promoting DC maturation and cytokine production. weeks old cotton rats were obtained from an inbred colony maintained at Envigo USA .",
"Further characterisation of the recombinant cotton rat CD40L revealed its functional activities in promoting DC maturation and cytokine production. weeks old cotton rats were obtained from an inbred colony maintained at Envigo USA . All animal experiments were conducted in accordance with Institutional Care and Use Committee IACUC of Health Canada Ottawa Animal Care Committee which approved this study. The rats were housed 3 animals per cage in Allentown NexGen individually ventilated cages with free access to food and water. These cages provided a floor space of 142 in 2 / 916 cm 2 . Body weight and any sign of distress were monitored daily. If anything associated the animal health was observed, a full examination would be conducted.",
"Body weight and any sign of distress were monitored daily. If anything associated the animal health was observed, a full examination would be conducted. As In this study spleen cells from normal, healthy animals were isolated, we did not observe any adverse reaction. To isolate splenocytes from the animals, isoflourane was used to put the animals to sleep via inhalation with oxygen for euthanasia. The spleens from three naïve cotton rats were removed aseptically and snap frozen in liquid nitrogen. The spleens were homogenized individually with a TissueLyser II Qiagen and total RNA extracted using the RNeasy Mini kit Qiagen with on-column DNase digestion according to the user's manual.",
"The spleens from three naïve cotton rats were removed aseptically and snap frozen in liquid nitrogen. The spleens were homogenized individually with a TissueLyser II Qiagen and total RNA extracted using the RNeasy Mini kit Qiagen with on-column DNase digestion according to the user's manual. The 3' RACE system Life Technologies was then used with to amplify the 3' portion of the cotton rat CD40L from the total RNA according to the manufacturer's instructions. A schematic of the 3' RACE procedure used is provided in S1 Fig. A gene specific primer 5'-GGACTCTATTATGTCTACACCCAAGTCACCTTCTG -3' was derived from a consensus sequence aligning the rat Rattus norvegicus UniProt: Q9Z2V2 , mouse Mus musculus UniProt: P27548 , and golden hamster Mesocricetus auratus NCBI Reference Sequence: XM_005084522.3 CD40L sequences obtained from the National Center for Biotechnology Information NCBI . Following first strand cDNA synthesis, the 3' portion of the cotton rat CD40L mRNA was PCR amplified using the consensus sequence derived gene specific primer and the abridged universal amplification primer with an annealing temperature at 56˚C.",
"A gene specific primer 5'-GGACTCTATTATGTCTACACCCAAGTCACCTTCTG -3' was derived from a consensus sequence aligning the rat Rattus norvegicus UniProt: Q9Z2V2 , mouse Mus musculus UniProt: P27548 , and golden hamster Mesocricetus auratus NCBI Reference Sequence: XM_005084522.3 CD40L sequences obtained from the National Center for Biotechnology Information NCBI . Following first strand cDNA synthesis, the 3' portion of the cotton rat CD40L mRNA was PCR amplified using the consensus sequence derived gene specific primer and the abridged universal amplification primer with an annealing temperature at 56˚C. The reverse complementary sequence of this primer was then used as a reverse primer with the forward primer 5'-GATAGAAACATACAGCCAACCTTCTCCCAGATC -3' to amplify the 5' portion of the cotton rat CD40L mRNA with an annealing temperature of 57˚C. All amplified fragments were sequenced with BigDye Terminator v.3.1 Cycle Sequencing kit ThermoFisher cat # 4336917 . Briefly, samples were amplified in a PTC-200 thermal cycle MJ Research with the following program: 26 cycles of 1˚C/S to 96˚C, 96˚C for 10 seconds, 1˚C/S to 50˚C, 50˚C for 5 seconds, 1˚C/S to 60˚C, 60˚C for 4 minutes. The samples were cleaned using DyeEx 2.0 Spin kit Qiagen cat # 63204 and loaded onto a 3130xl Genetic Analyzer Applied Biosystems .",
"Briefly, samples were amplified in a PTC-200 thermal cycle MJ Research with the following program: 26 cycles of 1˚C/S to 96˚C, 96˚C for 10 seconds, 1˚C/S to 50˚C, 50˚C for 5 seconds, 1˚C/S to 60˚C, 60˚C for 4 minutes. The samples were cleaned using DyeEx 2.0 Spin kit Qiagen cat # 63204 and loaded onto a 3130xl Genetic Analyzer Applied Biosystems . Raw sequencing data was edited by the instrument's software ThermoFisher 3130xl Genetic Analyzer Data Collection Software v3.0 , and then imported into GeneCodes Sequencher v4.6.1 sequencing analysis software for further editing. The final sequenced contigs are then imported to NCBI BLAST cgi to confirm the identity. Putative conserved domains, trimer interface, and receptor binding sites were determined by performing a standard protein BLAST blastp algorithm; The sequences producing significant alignments were imported into Geneosis software, Auckland, New Zealand . Multiple alignment was conducted as previously described , with phylogenetic analysis using Geneosis Pro 5.6.7.",
"Putative conserved domains, trimer interface, and receptor binding sites were determined by performing a standard protein BLAST blastp algorithm; The sequences producing significant alignments were imported into Geneosis software, Auckland, New Zealand . Multiple alignment was conducted as previously described , with phylogenetic analysis using Geneosis Pro 5.6.7. Once the mRNA sequence was confirmed, a construct was designed beginning with a kozak sequence 5'-CACCGCCGCCACC-3' , followed by a secretion signal consisting of 23 amino acid aa MLLAVLYCLLWSFQTSAGHFPRA from the human tyrosinase signal peptide as previously described . This is followed by six histidine residues to facilitate protein purification. Following this sequence, a 27-aa fragment from the bacteriophage T4 fibritin trimerization motif was added and finally connected to the full length 783bp open reading frame ORF of the cotton rat CD40L sequence at the C terminus. This construct was synthesized and cloned into pUC57 Biobasic, Markham, ON .",
"Following this sequence, a 27-aa fragment from the bacteriophage T4 fibritin trimerization motif was added and finally connected to the full length 783bp open reading frame ORF of the cotton rat CD40L sequence at the C terminus. This construct was synthesized and cloned into pUC57 Biobasic, Markham, ON . Generation of a recombinant vaccinia virus expressing cotton rat CD40L protein construct was achieved using a vaccinia virus E3L and K3L double deletion mutant virus as the parental virus and taterapoxvirus K3L as the positive selection marker Jingxin Cao, unpublished information . Briefly, the recombination plasmid vector for expression of the CD40L construct gene consists of the homologous flanking vaccinia DNA sequences targeting vaccinia A45R gene SOD homolog ; the CD40L construct gene driven by a modified vaccinia H5 promoter Vaccine 1996, 14:1451 , and taterapoxvirus 037 gene driven by vaccinia K3L promoter as the positive selection marker. The recombination vector was transfected into a HeLa PKR knockout cells infected with a vaccinia virus with both E3L and K3L genes deleted. Selection and purification of the recombinant vaccinia virus expressing the CD40L was done in BHK21 cells.",
"The recombination vector was transfected into a HeLa PKR knockout cells infected with a vaccinia virus with both E3L and K3L genes deleted. Selection and purification of the recombinant vaccinia virus expressing the CD40L was done in BHK21 cells. Expression of the CD40L protein was confirmed by Western blotting using His-tag Ab. Cell monolayers were lysed in sample buffer and homogenized using QIAshredder columns Qiagen . Western blotting was performed using 4 to 15% TGX gel and Tris/Glycine/SDS running buffer Bio-Rad Laboratories Inc. , and the protein samples were transferred to Immobilon-FL PVDF membranes Millipore . Protein was detected with Tetra-HIS Ab Qiagen and goat anti-mouse IRDye-800CW LiCor . Membranes were developed using the Odyssey system LiCor .",
"Protein was detected with Tetra-HIS Ab Qiagen and goat anti-mouse IRDye-800CW LiCor . Membranes were developed using the Odyssey system LiCor . The vaccinia virus carrying the crCD40L gene was propagated in BHK21 cells. The cells were collected and washed with PBS once and then lysed with a denaturing buffer 10 mM Tris-HCl, 100 mM sodium phosphate, 6 M guanidine hydrochloride, 10 mM reduced glutathione, pH 8.0 and disrupted by sonication on ice using a Branson sonifier 150 ThermoFisher, Waltham, MA at level 1 for two 10sec bursts with 1min rest on ice between. After separation of cell debris, the supernatant was added to a slurry of Ni-NTA resin Qiagen, Mississauga, ON, Canada 10 mL resin bed and stirred at room temperature for 30 min before loading into a column. The column was purified using an AKTA purifier Amersham Biosciences with Unicorn 5.3 software Amersham Biosciences .",
"After separation of cell debris, the supernatant was added to a slurry of Ni-NTA resin Qiagen, Mississauga, ON, Canada 10 mL resin bed and stirred at room temperature for 30 min before loading into a column. The column was purified using an AKTA purifier Amersham Biosciences with Unicorn 5.3 software Amersham Biosciences . Refolding was accomplished under oxidative conditions with a gradient of denaturing buffer to buffer B buffer B: 10 mM Tris-HCl, 100 mM sodium phosphate, pH 7.8 over 10 column volumes CVs . The column was then washed with three CVs of buffer B + 60 mM imidazole pH 7.8 to remove unspecific binding. The protein was eluted off the column with buffer B + 250 mM imidazole pH 7.8 . The resulting protein was dialysed against PBS pH 7.5 and then confirmed by western blot.",
"The protein was eluted off the column with buffer B + 250 mM imidazole pH 7.8 . The resulting protein was dialysed against PBS pH 7.5 and then confirmed by western blot. 96-well plates were coated with either recombinant mouse CD40L R&D Systems or the recombinant crCD40L protein 2ug/ml in 100μl PBS. Plates were washed with wash buffer PBS-0.1% tween-20 and then blocked with 200μl/well blocking buffer PBS containing 0.1% Tween 20 and 3%IgG Free BSA for 1 hour at 37˚C. Plates were washed with wash buffer and incubated at 37˚C for 1 hour with 100μl/well goat anti-mouseCD40L R&D Systems 2ug/ml in blocking buffer. Plates were subsequently washed and incubated at 37˚C for 1 hour with 100μl/well with rabbit anti-goat IgG HRP conjugate Zymed .",
"Plates were washed with wash buffer and incubated at 37˚C for 1 hour with 100μl/well goat anti-mouseCD40L R&D Systems 2ug/ml in blocking buffer. Plates were subsequently washed and incubated at 37˚C for 1 hour with 100μl/well with rabbit anti-goat IgG HRP conjugate Zymed . Plates were washed again and incubated for 10 min in the dark with 100μl/well 3,3'5,5'-tetramethylbenzidine substrate New England Bio Labs . The reaction was stopped with Stop solution New England Bio Labs and absorbance was read at 450nm on a BioTek Synergy 2 plate reader. Primary bone marrow cells from Balb/c mice Chicago, IL were thawed and cultured in dendritic cell medium from manufacture Cell Biologics M7711 supplemented with GMCSF Cell Biologics without IL-4 at 4x10 5 cells/well in a volume of 200μl. The cells were treated with 0.5μg/ml recombinant mouse CD40L Preprotech, Montreal, QC or the recombinant crCD40L protein at 0.5μg/ml, 5μg/ml, or 50μg/ml.",
"Primary bone marrow cells from Balb/c mice Chicago, IL were thawed and cultured in dendritic cell medium from manufacture Cell Biologics M7711 supplemented with GMCSF Cell Biologics without IL-4 at 4x10 5 cells/well in a volume of 200μl. The cells were treated with 0.5μg/ml recombinant mouse CD40L Preprotech, Montreal, QC or the recombinant crCD40L protein at 0.5μg/ml, 5μg/ml, or 50μg/ml. Forty hours later, flow cytometry was performed on a BD LSRFortessa cell analyser after 2 x 10 5 cells/tube were stained using CD11c-PE-CF594, CD54-FITC, CD40-BV786, CD80-BV421, and CD86-BV711 antibodies. All antibodies were purchased from BD Biosciences. The resulting spectra were analysed using FACS-Diva version 8.0.1 software. To assess IL-6 mRNA production of immature bone marrow murine DCs in response to targeting by recombinant crCD40L, quantitative real-time PCR was conducted on an ABI Prism 7500 Fast Sequence detection system Applied Biosystems .",
"The resulting spectra were analysed using FACS-Diva version 8.0.1 software. To assess IL-6 mRNA production of immature bone marrow murine DCs in response to targeting by recombinant crCD40L, quantitative real-time PCR was conducted on an ABI Prism 7500 Fast Sequence detection system Applied Biosystems . TaqMan assay reagent kits Applied Biosystems were used that contain pre-standardized primers and TaqMan MGB probes for IL-6 and 18S which were used to normalize the data. Total RNA was isolated from 8x10 5 stimulated bone marrow DCs using the RNeasy Mini Kit Qiagen according to manufactures instructions. The isolated RNA was used to make cDNA using the Superscript III First-Strand Synthesis System for RT-PCR Invitrogen according to manufacturer's instructions. The cDNA was then subjected to quantitative PCR using the TaqMan Fast Advanced Master Mix Applied Biosystems according to manufactures instructions.",
"The isolated RNA was used to make cDNA using the Superscript III First-Strand Synthesis System for RT-PCR Invitrogen according to manufacturer's instructions. The cDNA was then subjected to quantitative PCR using the TaqMan Fast Advanced Master Mix Applied Biosystems according to manufactures instructions. Samples were run in duplicate and C t values were obtained. Fold change over unstimulated DCs was calculated using the 2 -ΔΔCT method of relative quantification , using 18S as the housekeeping reference gene. To investigate IL-6 secretion by murine bone marrow DCs, supernatant from forty hour stimulated cultures were collected and assayed using the Mouse IL-6 DuoSet ELISA Kit R & D Systems following the manufacturer's protocol. The complete mRNA sequence of CD40L was obtained in two steps Fig 1 .",
"To investigate IL-6 secretion by murine bone marrow DCs, supernatant from forty hour stimulated cultures were collected and assayed using the Mouse IL-6 DuoSet ELISA Kit R & D Systems following the manufacturer's protocol. The complete mRNA sequence of CD40L was obtained in two steps Fig 1 . A sequence corresponding to nucleotides 535 through to the poly-A tail was obtained using the 3' RACE kit and mRNA as starting material, which was isolated from cotton rat splenocytes and a rodent consensus sequence as a primer. This portion of the sequence has the 3' un-translated region of the mRNA as well as the stop codon. The 5' end of the protein was obtained in the next step by PCR amplification of the cDNA obtained in the first step with the 3' RACE kit and the reverse complement of the consensus sequence primer and a second consensus sequence primer designed to bind to the beginning of the CD40L mRNA. The 783bp ORF encodes 260aa followed by a stop codon.",
"The 5' end of the protein was obtained in the next step by PCR amplification of the cDNA obtained in the first step with the 3' RACE kit and the reverse complement of the consensus sequence primer and a second consensus sequence primer designed to bind to the beginning of the CD40L mRNA. The 783bp ORF encodes 260aa followed by a stop codon. Comparison of the sequenced CD40L gene revealed that the crCD40L coding sequence shares 93%, 89%, and 83%, identity with golden hamster, rat, and mouse, respectively. At the amino acid aa level, the corresponding identities are 91%, 82%, and 82%, Fig 2a. At both the mRNA and aa levels, the crCD40L shared the closest similarity with Peromyscus maniculatus bairdii or deer mouse at 93% and 92% respectively. When sequence homology analysis is performed, crCD40L clusters with other members of the Cricetidae family Fig 2b.",
"At both the mRNA and aa levels, the crCD40L shared the closest similarity with Peromyscus maniculatus bairdii or deer mouse at 93% and 92% respectively. When sequence homology analysis is performed, crCD40L clusters with other members of the Cricetidae family Fig 2b. We next examined the functional domains in crCD40L in comparison with other known CD40L. As shown in Fig 3a, crCD40L has a putative tumor necrosis factor TNF superfamily Using EZmol software , we predicted folding of the protein as shown in Fig 3b. The cotton rat CD40L cDNA that we have isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783bp which coded for a 260 aa protein. The homology of cotton rat CD40L, at both the amino acid and nucleic acid level, is closer to members of the Cricetidae family hamster and deer mouse than to those of the Muridae family rat and mouse as shown in Fig 2b.",
"The cotton rat CD40L cDNA that we have isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783bp which coded for a 260 aa protein. The homology of cotton rat CD40L, at both the amino acid and nucleic acid level, is closer to members of the Cricetidae family hamster and deer mouse than to those of the Muridae family rat and mouse as shown in Fig 2b. As with other known CD40L proteins, there is a putative TNF superfamily domain, a transmembrane domain, trimerization sites, and receptor binding sites . TNF superfamily members include TNF TNF-alpha , LT lymphotoxin-alpha, TNF-beta , CD40 ligand, Apo2L TRAIL , Fas ligand, and osteoprotegerin OPG ligand, among others . The TNF superfamily is composed of 19 ligands and 29 receptors, in which each has vastly diversified roles in the body and exhibit pro-inflammatory activity, partly via activation of NF-kB . Members of this family generally have an intracellular N-terminal domain, a short transmembrane segment, an extracellular stalk, and a globular TNF-like extracellular domain of about 150 residues .",
"The TNF superfamily is composed of 19 ligands and 29 receptors, in which each has vastly diversified roles in the body and exhibit pro-inflammatory activity, partly via activation of NF-kB . Members of this family generally have an intracellular N-terminal domain, a short transmembrane segment, an extracellular stalk, and a globular TNF-like extracellular domain of about 150 residues . They initiate apoptosis by binding to related receptors, some of which have intracellular death domains . These proteins typically form homo-or hetero-trimeric complexes and bind one elongated receptor molecule along each of three clefts formed by neighboring monomers of the trimer and ligand trimerization is for receptor binding . All seven known conserved residues that constitute the trimer interface on the conserved TNF domain , were mapped to the putative crCD40L protein sequence. Additionally, all six known conserved receptor binding sites on the conserved TNF domain , were mapped to the crCD40L protein sequence.",
"All seven known conserved residues that constitute the trimer interface on the conserved TNF domain , were mapped to the putative crCD40L protein sequence. Additionally, all six known conserved receptor binding sites on the conserved TNF domain , were mapped to the crCD40L protein sequence. In order to further evaluate the crCD40L deduced sequence, the full 783bp ORF of the crCD40L was cloned into a vaccinia virus vector. The crCD40L construct was designed to carry a secretion signal, histidine tag, and a trimerization motif Fig 4a . Selection and purification of the recombinant vaccinia virus expressing the CD40L construct was conducted in BHK21 cells. Western blot with anti-histidine antibody Ab was used to confirm expression of the CD40L protein construct Fig 4b and S2 Fig.",
"Selection and purification of the recombinant vaccinia virus expressing the CD40L construct was conducted in BHK21 cells. Western blot with anti-histidine antibody Ab was used to confirm expression of the CD40L protein construct Fig 4b and S2 Fig. The resulting 36 kDa protein product was found in both the cell lysate and supernatant faint band-48 hours only . Since the highest expression was found in the cell lysate, it was used for further purification of the protein. It should be noted that the protein was only able to be detected under reducing conditions. Under non-reducing conditions, the protein was unable to be detected by the anti-histidine Ab, even in the cell lysate data not shown .",
"It should be noted that the protein was only able to be detected under reducing conditions. Under non-reducing conditions, the protein was unable to be detected by the anti-histidine Ab, even in the cell lysate data not shown . This indicates that the histidine tag is folded within the trimer and is unavailable in the native form for purification. This is an additional reason for the need to purify the protein from the cell lysate under harsh denaturing conditions followed by protein refolding. The reason we utilized a mammalian expression system to produce the protein rather than a bacterial system is to facilitate its proper folding into its native structure, trimerization, and glycosylation. The aa backbone predicts a protein of 29 kDa, yet initial studies of the CD40L protein suggested a molecular mass of 39 kDa, and on most cell types the molecular mass of CD40L is 32-33kDa, consistent with extensive post-translation modification .",
"The reason we utilized a mammalian expression system to produce the protein rather than a bacterial system is to facilitate its proper folding into its native structure, trimerization, and glycosylation. The aa backbone predicts a protein of 29 kDa, yet initial studies of the CD40L protein suggested a molecular mass of 39 kDa, and on most cell types the molecular mass of CD40L is 32-33kDa, consistent with extensive post-translation modification . The BHK21 cells expressing the crCD40L construct were collected and lysed with 6 M guanidine hydrochloride with reduced glutathione and sonication. The lysate was loaded on the nickel column and the washed with denaturing buffer as described in materials and methods. The bound proteins were refolded on the column with gradient buffer exchange, to allow slow refold the protein, given that CD40L biological activity is dependent on a homo-trimer configuration . The resulting bound protein was subsequently eluted with imidazole.",
"The bound proteins were refolded on the column with gradient buffer exchange, to allow slow refold the protein, given that CD40L biological activity is dependent on a homo-trimer configuration . The resulting bound protein was subsequently eluted with imidazole. The resulting fractions that showed a peak were pooled and dialysed against PBS. The purified protein was confirmed in ELISA. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence, an Ab known to detect mouse CD40L was used to identify the purified crCD40L protein. The purified recombinant crCD40L was used as a coating antigen in a concentration gradient manner, and was detected with an Ab generated against the mouse CD40L at all concentrations Fig 5 .",
"Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence, an Ab known to detect mouse CD40L was used to identify the purified crCD40L protein. The purified recombinant crCD40L was used as a coating antigen in a concentration gradient manner, and was detected with an Ab generated against the mouse CD40L at all concentrations Fig 5 . Uncoated controls were performed in parallel and were negative for CD40L in ELISA. We measured the overall strength of the antigen-antibody complex in the presence of 6M urea . The avidity of the cotton rat CD40L for the anti-mouse CD40L antibody was decreased in the presence of 6M urea at all concentrations. Clearly, as the antibody used was raised against mouse CD40L, the crCD40L is detected by mouse CD40L.",
"The avidity of the cotton rat CD40L for the anti-mouse CD40L antibody was decreased in the presence of 6M urea at all concentrations. Clearly, as the antibody used was raised against mouse CD40L, the crCD40L is detected by mouse CD40L. crCD40L was expressed in vaccinia virus and purified from infected BHK21 cell lysate on a nickel column. The purified protein was detected by ELISA using a mouse antibody against CD40L in a concentration gradient dependent manner. The avidity of the mouse CD40L antibody to the cotton rat CD40L protein was evaluated in the presence of 6M urea. The difference between the untreated and 6M urea treated for each group was calculated using students t-test ÃÃÃ p<0.001, ÃÃÃÃ p<0.0001 n = 2 .",
"The avidity of the mouse CD40L antibody to the cotton rat CD40L protein was evaluated in the presence of 6M urea. The difference between the untreated and 6M urea treated for each group was calculated using students t-test ÃÃÃ p<0.001, ÃÃÃÃ p<0.0001 n = 2 . Data shown is a representative experiment of three separate experiments where two n = 2 technical replicates are conducted in each experiment. The no-coating and noprimary antibody negative controls gave average OD values of 0.56 and 0.107 respectively. addition of urea treatment would substantially weaken the interaction between the antibody and crCD40L. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence with similar functional domains, we evaluated the biological activity of the recombinant crCD40L on immature murine bone marrow DCs.",
"addition of urea treatment would substantially weaken the interaction between the antibody and crCD40L. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence with similar functional domains, we evaluated the biological activity of the recombinant crCD40L on immature murine bone marrow DCs. We conducted experiments based on known functional activities of CD40L in other animal species. Specifically, maturation of immature DCs after exposure to antigen is known to play a crucial role in their immunity-stimulating function , while trimeric recombinant CD40L has been shown to stimulate DC immunomodulating functions . When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated .",
"When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L . Moreover, mouse I-A d major histocompatibility complex is also up-regulated upon stimulation with CD40L . When our recombinant crCD40L was used to stimulate immature murine bone marrow DCs, we observed similar results to that when murine CD40L is used Tables 1 and 2 . CD11c was down regulated in both median flouresence intensity Table 1 and the percentage of positive cells Table 2 .",
"When our recombinant crCD40L was used to stimulate immature murine bone marrow DCs, we observed similar results to that when murine CD40L is used Tables 1 and 2 . CD11c was down regulated in both median flouresence intensity Table 1 and the percentage of positive cells Table 2 . The co-stimulatory molecules CD54, CD40, CD80, and CD86 were all up-regulated in both median fluorescence intensity Table 1 and the percentage of positive cells Table 2 . The Mouse I-A d major histocompatibility complex was upregulated in median fluorescence intensity Table 1 but not up-regulated in terms of the overall percentage of positive cells Table 2 . We speculate this to be due to the species incompatibility since we are stimulating mouse bone marrow cells with cotton rat CD40L. Nevertheless, the crCD40L was able to promote up-regulation of key co-stimulatory markers on immature DCs promoting DC maturation.",
"We speculate this to be due to the species incompatibility since we are stimulating mouse bone marrow cells with cotton rat CD40L. Nevertheless, the crCD40L was able to promote up-regulation of key co-stimulatory markers on immature DCs promoting DC maturation. The gating strategy used for the flow cytometry analysis is provided in S3 Fig along with overlapping histograms of the intracellular adhesion marker and co-stimulatory markers. CD40-induced activation of cytokine gene expression in DCs by CD40L is an important process in the initiation of primary immune responses and is critical for DC maturation and the generation of antigen-specific T cell responses . IL-6 is a highly pleiotropic cytokine in that it stimulates the activation, proliferation, and survival of T cells, and furthermore, modifies DC function and survival . We tested if the recombinant crCD40L could induce IL-6 gene expression Fig 6a and production of the cytokine Fig 6b by immature murine bone marrow DCs.",
"IL-6 is a highly pleiotropic cytokine in that it stimulates the activation, proliferation, and survival of T cells, and furthermore, modifies DC function and survival . We tested if the recombinant crCD40L could induce IL-6 gene expression Fig 6a and production of the cytokine Fig 6b by immature murine bone marrow DCs. The results indicate that a significant increase in both IL-6 gene expression and cytokine production in immature murine bone marrow DCs was observed forty hours after stimulation with the crCD40L. Collectively, the observation that both the upregulation of immature DC cell surface maturation markers and increased IL-6 gene expression and cytokine production provide strong evidence of the biological activity of crCD40L. In summary, the cotton rat CD40L cDNA that we isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783 bp which coded for a 260 aa protein. The recombinant cotton rat CD40L was recognized by an Ab against mouse CD40L in direct ELISA, and showed biological activity by upregulating maturation markers CD40, CD54, CD80, and CD86 as well as I-A d on immature bone marrow murine DCs and moreover, inducing upregulation of IL-6 gene and cytokine expression in these cells.",
"In summary, the cotton rat CD40L cDNA that we isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783 bp which coded for a 260 aa protein. The recombinant cotton rat CD40L was recognized by an Ab against mouse CD40L in direct ELISA, and showed biological activity by upregulating maturation markers CD40, CD54, CD80, and CD86 as well as I-A d on immature bone marrow murine DCs and moreover, inducing upregulation of IL-6 gene and cytokine expression in these cells. The isolation of the cotton rat CD40L sequence and availability of CD40L has the potential to positively impact basic immunological research and vaccine development, given the critical importance of this protein in orchestrating immune responses ."
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What is the structure of the CD40 Ligand?
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a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet
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[
"Cotton rats are an important animal model to study infectious diseases. They have demonstrated higher susceptibility to a wider variety of human pathogens than other rodents and are also the animal model of choice for pre-clinical evaluations of some vaccine candidates. However, the genome of cotton rats remains to be fully sequenced, with much fewer genes cloned and characterised compared to other rodent species. Here we report the cloning and characterization of CD40 ligand, whose human and murine counterparts are known to be expressed on a range of cell types including activated T cells and B cells, dendritic cells, granulocytes, macrophages and platelets and exerts a broad array of immune responses. The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame ORF of 783bp coding for a 260 amino acid protein. The recombinant cotton rat CD40L protein was recognized by an antibody against mouse CD40L.",
"The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame ORF of 783bp coding for a 260 amino acid protein. The recombinant cotton rat CD40L protein was recognized by an antibody against mouse CD40L. Moreover, it demonstrated functional activities on immature bone marrow dendritic cells by upregulating surface maturation markers CD40, CD54, CD80, and CD86 , and increasing IL-6 gene and protein expression. The availability of CD40L gene identity could greatly facilitate mechanistic research on pathogen-induced-immunopathogenesis and vaccine-elicited immune responses. Text: The cotton rat Sigmodon hispidus was first used in polio research in the 1930s , and throughout the last century, it has proven to be an excellent model for biomedical research . Historically in biomedical research, the mouse has been exploited as the default animal model.",
"Text: The cotton rat Sigmodon hispidus was first used in polio research in the 1930s , and throughout the last century, it has proven to be an excellent model for biomedical research . Historically in biomedical research, the mouse has been exploited as the default animal model. This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus . This model has been valuable for adenovirus-based gene replacement therapy research , and was also proven to be indispensable in pre-clinical evaluation of the prophylactic antibodies RespiGam 1 , and Synagis 1 .",
"On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus . This model has been valuable for adenovirus-based gene replacement therapy research , and was also proven to be indispensable in pre-clinical evaluation of the prophylactic antibodies RespiGam 1 , and Synagis 1 . Indeed, the cotton rat model was found to be valuable in terms of its biological and immunological relevance, it was deemed unnecessary to test the adenovirus-based gene therapy and the Synagis 1 prophylactic treatment against RSV disease in non-human primate prior to the human trials . A number of methods and reagents have been developed for the analysis of immune responses in cotton rats over the last decade. Up to date, more than 200 genes encoding cytokines, chemokines, cell surface markers and regulatory molecules have been cloned, with various related research reagents being commercially available. As a result, the use of cotton rats in pathogenesis studies addressing mechanistic questions has significantly increased.",
"Up to date, more than 200 genes encoding cytokines, chemokines, cell surface markers and regulatory molecules have been cloned, with various related research reagents being commercially available. As a result, the use of cotton rats in pathogenesis studies addressing mechanistic questions has significantly increased. Nevertheless, the gene encoding CD154 and CD40 ligand CD40L , remains elusive. CD40L plays a critical role in orchestrating immune responses against pathogens. Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands .",
"Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets . It has been shown that upon interacting with its receptor, CD40, CD40L induces profound effects on T cells, DCs, B cells, endothelial cells, as well as many cells of the hematopoietic and non-hematopoietic systems. Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin Ig isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy.",
"Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin Ig isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy. It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes . Here we report the successful cloning of the gene encoding cotton rat CD40L crCD40L ; we also expressed and purified the CD40L produced in mammalian cells. Further characterisation of the recombinant cotton rat CD40L revealed its functional activities in promoting DC maturation and cytokine production. weeks old cotton rats were obtained from an inbred colony maintained at Envigo USA .",
"Further characterisation of the recombinant cotton rat CD40L revealed its functional activities in promoting DC maturation and cytokine production. weeks old cotton rats were obtained from an inbred colony maintained at Envigo USA . All animal experiments were conducted in accordance with Institutional Care and Use Committee IACUC of Health Canada Ottawa Animal Care Committee which approved this study. The rats were housed 3 animals per cage in Allentown NexGen individually ventilated cages with free access to food and water. These cages provided a floor space of 142 in 2 / 916 cm 2 . Body weight and any sign of distress were monitored daily. If anything associated the animal health was observed, a full examination would be conducted.",
"Body weight and any sign of distress were monitored daily. If anything associated the animal health was observed, a full examination would be conducted. As In this study spleen cells from normal, healthy animals were isolated, we did not observe any adverse reaction. To isolate splenocytes from the animals, isoflourane was used to put the animals to sleep via inhalation with oxygen for euthanasia. The spleens from three naïve cotton rats were removed aseptically and snap frozen in liquid nitrogen. The spleens were homogenized individually with a TissueLyser II Qiagen and total RNA extracted using the RNeasy Mini kit Qiagen with on-column DNase digestion according to the user's manual.",
"The spleens from three naïve cotton rats were removed aseptically and snap frozen in liquid nitrogen. The spleens were homogenized individually with a TissueLyser II Qiagen and total RNA extracted using the RNeasy Mini kit Qiagen with on-column DNase digestion according to the user's manual. The 3' RACE system Life Technologies was then used with to amplify the 3' portion of the cotton rat CD40L from the total RNA according to the manufacturer's instructions. A schematic of the 3' RACE procedure used is provided in S1 Fig. A gene specific primer 5'-GGACTCTATTATGTCTACACCCAAGTCACCTTCTG -3' was derived from a consensus sequence aligning the rat Rattus norvegicus UniProt: Q9Z2V2 , mouse Mus musculus UniProt: P27548 , and golden hamster Mesocricetus auratus NCBI Reference Sequence: XM_005084522.3 CD40L sequences obtained from the National Center for Biotechnology Information NCBI . Following first strand cDNA synthesis, the 3' portion of the cotton rat CD40L mRNA was PCR amplified using the consensus sequence derived gene specific primer and the abridged universal amplification primer with an annealing temperature at 56˚C.",
"A gene specific primer 5'-GGACTCTATTATGTCTACACCCAAGTCACCTTCTG -3' was derived from a consensus sequence aligning the rat Rattus norvegicus UniProt: Q9Z2V2 , mouse Mus musculus UniProt: P27548 , and golden hamster Mesocricetus auratus NCBI Reference Sequence: XM_005084522.3 CD40L sequences obtained from the National Center for Biotechnology Information NCBI . Following first strand cDNA synthesis, the 3' portion of the cotton rat CD40L mRNA was PCR amplified using the consensus sequence derived gene specific primer and the abridged universal amplification primer with an annealing temperature at 56˚C. The reverse complementary sequence of this primer was then used as a reverse primer with the forward primer 5'-GATAGAAACATACAGCCAACCTTCTCCCAGATC -3' to amplify the 5' portion of the cotton rat CD40L mRNA with an annealing temperature of 57˚C. All amplified fragments were sequenced with BigDye Terminator v.3.1 Cycle Sequencing kit ThermoFisher cat # 4336917 . Briefly, samples were amplified in a PTC-200 thermal cycle MJ Research with the following program: 26 cycles of 1˚C/S to 96˚C, 96˚C for 10 seconds, 1˚C/S to 50˚C, 50˚C for 5 seconds, 1˚C/S to 60˚C, 60˚C for 4 minutes. The samples were cleaned using DyeEx 2.0 Spin kit Qiagen cat # 63204 and loaded onto a 3130xl Genetic Analyzer Applied Biosystems .",
"Briefly, samples were amplified in a PTC-200 thermal cycle MJ Research with the following program: 26 cycles of 1˚C/S to 96˚C, 96˚C for 10 seconds, 1˚C/S to 50˚C, 50˚C for 5 seconds, 1˚C/S to 60˚C, 60˚C for 4 minutes. The samples were cleaned using DyeEx 2.0 Spin kit Qiagen cat # 63204 and loaded onto a 3130xl Genetic Analyzer Applied Biosystems . Raw sequencing data was edited by the instrument's software ThermoFisher 3130xl Genetic Analyzer Data Collection Software v3.0 , and then imported into GeneCodes Sequencher v4.6.1 sequencing analysis software for further editing. The final sequenced contigs are then imported to NCBI BLAST cgi to confirm the identity. Putative conserved domains, trimer interface, and receptor binding sites were determined by performing a standard protein BLAST blastp algorithm; The sequences producing significant alignments were imported into Geneosis software, Auckland, New Zealand . Multiple alignment was conducted as previously described , with phylogenetic analysis using Geneosis Pro 5.6.7.",
"Putative conserved domains, trimer interface, and receptor binding sites were determined by performing a standard protein BLAST blastp algorithm; The sequences producing significant alignments were imported into Geneosis software, Auckland, New Zealand . Multiple alignment was conducted as previously described , with phylogenetic analysis using Geneosis Pro 5.6.7. Once the mRNA sequence was confirmed, a construct was designed beginning with a kozak sequence 5'-CACCGCCGCCACC-3' , followed by a secretion signal consisting of 23 amino acid aa MLLAVLYCLLWSFQTSAGHFPRA from the human tyrosinase signal peptide as previously described . This is followed by six histidine residues to facilitate protein purification. Following this sequence, a 27-aa fragment from the bacteriophage T4 fibritin trimerization motif was added and finally connected to the full length 783bp open reading frame ORF of the cotton rat CD40L sequence at the C terminus. This construct was synthesized and cloned into pUC57 Biobasic, Markham, ON .",
"Following this sequence, a 27-aa fragment from the bacteriophage T4 fibritin trimerization motif was added and finally connected to the full length 783bp open reading frame ORF of the cotton rat CD40L sequence at the C terminus. This construct was synthesized and cloned into pUC57 Biobasic, Markham, ON . Generation of a recombinant vaccinia virus expressing cotton rat CD40L protein construct was achieved using a vaccinia virus E3L and K3L double deletion mutant virus as the parental virus and taterapoxvirus K3L as the positive selection marker Jingxin Cao, unpublished information . Briefly, the recombination plasmid vector for expression of the CD40L construct gene consists of the homologous flanking vaccinia DNA sequences targeting vaccinia A45R gene SOD homolog ; the CD40L construct gene driven by a modified vaccinia H5 promoter Vaccine 1996, 14:1451 , and taterapoxvirus 037 gene driven by vaccinia K3L promoter as the positive selection marker. The recombination vector was transfected into a HeLa PKR knockout cells infected with a vaccinia virus with both E3L and K3L genes deleted. Selection and purification of the recombinant vaccinia virus expressing the CD40L was done in BHK21 cells.",
"The recombination vector was transfected into a HeLa PKR knockout cells infected with a vaccinia virus with both E3L and K3L genes deleted. Selection and purification of the recombinant vaccinia virus expressing the CD40L was done in BHK21 cells. Expression of the CD40L protein was confirmed by Western blotting using His-tag Ab. Cell monolayers were lysed in sample buffer and homogenized using QIAshredder columns Qiagen . Western blotting was performed using 4 to 15% TGX gel and Tris/Glycine/SDS running buffer Bio-Rad Laboratories Inc. , and the protein samples were transferred to Immobilon-FL PVDF membranes Millipore . Protein was detected with Tetra-HIS Ab Qiagen and goat anti-mouse IRDye-800CW LiCor . Membranes were developed using the Odyssey system LiCor .",
"Protein was detected with Tetra-HIS Ab Qiagen and goat anti-mouse IRDye-800CW LiCor . Membranes were developed using the Odyssey system LiCor . The vaccinia virus carrying the crCD40L gene was propagated in BHK21 cells. The cells were collected and washed with PBS once and then lysed with a denaturing buffer 10 mM Tris-HCl, 100 mM sodium phosphate, 6 M guanidine hydrochloride, 10 mM reduced glutathione, pH 8.0 and disrupted by sonication on ice using a Branson sonifier 150 ThermoFisher, Waltham, MA at level 1 for two 10sec bursts with 1min rest on ice between. After separation of cell debris, the supernatant was added to a slurry of Ni-NTA resin Qiagen, Mississauga, ON, Canada 10 mL resin bed and stirred at room temperature for 30 min before loading into a column. The column was purified using an AKTA purifier Amersham Biosciences with Unicorn 5.3 software Amersham Biosciences .",
"After separation of cell debris, the supernatant was added to a slurry of Ni-NTA resin Qiagen, Mississauga, ON, Canada 10 mL resin bed and stirred at room temperature for 30 min before loading into a column. The column was purified using an AKTA purifier Amersham Biosciences with Unicorn 5.3 software Amersham Biosciences . Refolding was accomplished under oxidative conditions with a gradient of denaturing buffer to buffer B buffer B: 10 mM Tris-HCl, 100 mM sodium phosphate, pH 7.8 over 10 column volumes CVs . The column was then washed with three CVs of buffer B + 60 mM imidazole pH 7.8 to remove unspecific binding. The protein was eluted off the column with buffer B + 250 mM imidazole pH 7.8 . The resulting protein was dialysed against PBS pH 7.5 and then confirmed by western blot.",
"The protein was eluted off the column with buffer B + 250 mM imidazole pH 7.8 . The resulting protein was dialysed against PBS pH 7.5 and then confirmed by western blot. 96-well plates were coated with either recombinant mouse CD40L R&D Systems or the recombinant crCD40L protein 2ug/ml in 100μl PBS. Plates were washed with wash buffer PBS-0.1% tween-20 and then blocked with 200μl/well blocking buffer PBS containing 0.1% Tween 20 and 3%IgG Free BSA for 1 hour at 37˚C. Plates were washed with wash buffer and incubated at 37˚C for 1 hour with 100μl/well goat anti-mouseCD40L R&D Systems 2ug/ml in blocking buffer. Plates were subsequently washed and incubated at 37˚C for 1 hour with 100μl/well with rabbit anti-goat IgG HRP conjugate Zymed .",
"Plates were washed with wash buffer and incubated at 37˚C for 1 hour with 100μl/well goat anti-mouseCD40L R&D Systems 2ug/ml in blocking buffer. Plates were subsequently washed and incubated at 37˚C for 1 hour with 100μl/well with rabbit anti-goat IgG HRP conjugate Zymed . Plates were washed again and incubated for 10 min in the dark with 100μl/well 3,3'5,5'-tetramethylbenzidine substrate New England Bio Labs . The reaction was stopped with Stop solution New England Bio Labs and absorbance was read at 450nm on a BioTek Synergy 2 plate reader. Primary bone marrow cells from Balb/c mice Chicago, IL were thawed and cultured in dendritic cell medium from manufacture Cell Biologics M7711 supplemented with GMCSF Cell Biologics without IL-4 at 4x10 5 cells/well in a volume of 200μl. The cells were treated with 0.5μg/ml recombinant mouse CD40L Preprotech, Montreal, QC or the recombinant crCD40L protein at 0.5μg/ml, 5μg/ml, or 50μg/ml.",
"Primary bone marrow cells from Balb/c mice Chicago, IL were thawed and cultured in dendritic cell medium from manufacture Cell Biologics M7711 supplemented with GMCSF Cell Biologics without IL-4 at 4x10 5 cells/well in a volume of 200μl. The cells were treated with 0.5μg/ml recombinant mouse CD40L Preprotech, Montreal, QC or the recombinant crCD40L protein at 0.5μg/ml, 5μg/ml, or 50μg/ml. Forty hours later, flow cytometry was performed on a BD LSRFortessa cell analyser after 2 x 10 5 cells/tube were stained using CD11c-PE-CF594, CD54-FITC, CD40-BV786, CD80-BV421, and CD86-BV711 antibodies. All antibodies were purchased from BD Biosciences. The resulting spectra were analysed using FACS-Diva version 8.0.1 software. To assess IL-6 mRNA production of immature bone marrow murine DCs in response to targeting by recombinant crCD40L, quantitative real-time PCR was conducted on an ABI Prism 7500 Fast Sequence detection system Applied Biosystems .",
"The resulting spectra were analysed using FACS-Diva version 8.0.1 software. To assess IL-6 mRNA production of immature bone marrow murine DCs in response to targeting by recombinant crCD40L, quantitative real-time PCR was conducted on an ABI Prism 7500 Fast Sequence detection system Applied Biosystems . TaqMan assay reagent kits Applied Biosystems were used that contain pre-standardized primers and TaqMan MGB probes for IL-6 and 18S which were used to normalize the data. Total RNA was isolated from 8x10 5 stimulated bone marrow DCs using the RNeasy Mini Kit Qiagen according to manufactures instructions. The isolated RNA was used to make cDNA using the Superscript III First-Strand Synthesis System for RT-PCR Invitrogen according to manufacturer's instructions. The cDNA was then subjected to quantitative PCR using the TaqMan Fast Advanced Master Mix Applied Biosystems according to manufactures instructions.",
"The isolated RNA was used to make cDNA using the Superscript III First-Strand Synthesis System for RT-PCR Invitrogen according to manufacturer's instructions. The cDNA was then subjected to quantitative PCR using the TaqMan Fast Advanced Master Mix Applied Biosystems according to manufactures instructions. Samples were run in duplicate and C t values were obtained. Fold change over unstimulated DCs was calculated using the 2 -ΔΔCT method of relative quantification , using 18S as the housekeeping reference gene. To investigate IL-6 secretion by murine bone marrow DCs, supernatant from forty hour stimulated cultures were collected and assayed using the Mouse IL-6 DuoSet ELISA Kit R & D Systems following the manufacturer's protocol. The complete mRNA sequence of CD40L was obtained in two steps Fig 1 .",
"To investigate IL-6 secretion by murine bone marrow DCs, supernatant from forty hour stimulated cultures were collected and assayed using the Mouse IL-6 DuoSet ELISA Kit R & D Systems following the manufacturer's protocol. The complete mRNA sequence of CD40L was obtained in two steps Fig 1 . A sequence corresponding to nucleotides 535 through to the poly-A tail was obtained using the 3' RACE kit and mRNA as starting material, which was isolated from cotton rat splenocytes and a rodent consensus sequence as a primer. This portion of the sequence has the 3' un-translated region of the mRNA as well as the stop codon. The 5' end of the protein was obtained in the next step by PCR amplification of the cDNA obtained in the first step with the 3' RACE kit and the reverse complement of the consensus sequence primer and a second consensus sequence primer designed to bind to the beginning of the CD40L mRNA. The 783bp ORF encodes 260aa followed by a stop codon.",
"The 5' end of the protein was obtained in the next step by PCR amplification of the cDNA obtained in the first step with the 3' RACE kit and the reverse complement of the consensus sequence primer and a second consensus sequence primer designed to bind to the beginning of the CD40L mRNA. The 783bp ORF encodes 260aa followed by a stop codon. Comparison of the sequenced CD40L gene revealed that the crCD40L coding sequence shares 93%, 89%, and 83%, identity with golden hamster, rat, and mouse, respectively. At the amino acid aa level, the corresponding identities are 91%, 82%, and 82%, Fig 2a. At both the mRNA and aa levels, the crCD40L shared the closest similarity with Peromyscus maniculatus bairdii or deer mouse at 93% and 92% respectively. When sequence homology analysis is performed, crCD40L clusters with other members of the Cricetidae family Fig 2b.",
"At both the mRNA and aa levels, the crCD40L shared the closest similarity with Peromyscus maniculatus bairdii or deer mouse at 93% and 92% respectively. When sequence homology analysis is performed, crCD40L clusters with other members of the Cricetidae family Fig 2b. We next examined the functional domains in crCD40L in comparison with other known CD40L. As shown in Fig 3a, crCD40L has a putative tumor necrosis factor TNF superfamily Using EZmol software , we predicted folding of the protein as shown in Fig 3b. The cotton rat CD40L cDNA that we have isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783bp which coded for a 260 aa protein. The homology of cotton rat CD40L, at both the amino acid and nucleic acid level, is closer to members of the Cricetidae family hamster and deer mouse than to those of the Muridae family rat and mouse as shown in Fig 2b.",
"The cotton rat CD40L cDNA that we have isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783bp which coded for a 260 aa protein. The homology of cotton rat CD40L, at both the amino acid and nucleic acid level, is closer to members of the Cricetidae family hamster and deer mouse than to those of the Muridae family rat and mouse as shown in Fig 2b. As with other known CD40L proteins, there is a putative TNF superfamily domain, a transmembrane domain, trimerization sites, and receptor binding sites . TNF superfamily members include TNF TNF-alpha , LT lymphotoxin-alpha, TNF-beta , CD40 ligand, Apo2L TRAIL , Fas ligand, and osteoprotegerin OPG ligand, among others . The TNF superfamily is composed of 19 ligands and 29 receptors, in which each has vastly diversified roles in the body and exhibit pro-inflammatory activity, partly via activation of NF-kB . Members of this family generally have an intracellular N-terminal domain, a short transmembrane segment, an extracellular stalk, and a globular TNF-like extracellular domain of about 150 residues .",
"The TNF superfamily is composed of 19 ligands and 29 receptors, in which each has vastly diversified roles in the body and exhibit pro-inflammatory activity, partly via activation of NF-kB . Members of this family generally have an intracellular N-terminal domain, a short transmembrane segment, an extracellular stalk, and a globular TNF-like extracellular domain of about 150 residues . They initiate apoptosis by binding to related receptors, some of which have intracellular death domains . These proteins typically form homo-or hetero-trimeric complexes and bind one elongated receptor molecule along each of three clefts formed by neighboring monomers of the trimer and ligand trimerization is for receptor binding . All seven known conserved residues that constitute the trimer interface on the conserved TNF domain , were mapped to the putative crCD40L protein sequence. Additionally, all six known conserved receptor binding sites on the conserved TNF domain , were mapped to the crCD40L protein sequence.",
"All seven known conserved residues that constitute the trimer interface on the conserved TNF domain , were mapped to the putative crCD40L protein sequence. Additionally, all six known conserved receptor binding sites on the conserved TNF domain , were mapped to the crCD40L protein sequence. In order to further evaluate the crCD40L deduced sequence, the full 783bp ORF of the crCD40L was cloned into a vaccinia virus vector. The crCD40L construct was designed to carry a secretion signal, histidine tag, and a trimerization motif Fig 4a . Selection and purification of the recombinant vaccinia virus expressing the CD40L construct was conducted in BHK21 cells. Western blot with anti-histidine antibody Ab was used to confirm expression of the CD40L protein construct Fig 4b and S2 Fig.",
"Selection and purification of the recombinant vaccinia virus expressing the CD40L construct was conducted in BHK21 cells. Western blot with anti-histidine antibody Ab was used to confirm expression of the CD40L protein construct Fig 4b and S2 Fig. The resulting 36 kDa protein product was found in both the cell lysate and supernatant faint band-48 hours only . Since the highest expression was found in the cell lysate, it was used for further purification of the protein. It should be noted that the protein was only able to be detected under reducing conditions. Under non-reducing conditions, the protein was unable to be detected by the anti-histidine Ab, even in the cell lysate data not shown .",
"It should be noted that the protein was only able to be detected under reducing conditions. Under non-reducing conditions, the protein was unable to be detected by the anti-histidine Ab, even in the cell lysate data not shown . This indicates that the histidine tag is folded within the trimer and is unavailable in the native form for purification. This is an additional reason for the need to purify the protein from the cell lysate under harsh denaturing conditions followed by protein refolding. The reason we utilized a mammalian expression system to produce the protein rather than a bacterial system is to facilitate its proper folding into its native structure, trimerization, and glycosylation. The aa backbone predicts a protein of 29 kDa, yet initial studies of the CD40L protein suggested a molecular mass of 39 kDa, and on most cell types the molecular mass of CD40L is 32-33kDa, consistent with extensive post-translation modification .",
"The reason we utilized a mammalian expression system to produce the protein rather than a bacterial system is to facilitate its proper folding into its native structure, trimerization, and glycosylation. The aa backbone predicts a protein of 29 kDa, yet initial studies of the CD40L protein suggested a molecular mass of 39 kDa, and on most cell types the molecular mass of CD40L is 32-33kDa, consistent with extensive post-translation modification . The BHK21 cells expressing the crCD40L construct were collected and lysed with 6 M guanidine hydrochloride with reduced glutathione and sonication. The lysate was loaded on the nickel column and the washed with denaturing buffer as described in materials and methods. The bound proteins were refolded on the column with gradient buffer exchange, to allow slow refold the protein, given that CD40L biological activity is dependent on a homo-trimer configuration . The resulting bound protein was subsequently eluted with imidazole.",
"The bound proteins were refolded on the column with gradient buffer exchange, to allow slow refold the protein, given that CD40L biological activity is dependent on a homo-trimer configuration . The resulting bound protein was subsequently eluted with imidazole. The resulting fractions that showed a peak were pooled and dialysed against PBS. The purified protein was confirmed in ELISA. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence, an Ab known to detect mouse CD40L was used to identify the purified crCD40L protein. The purified recombinant crCD40L was used as a coating antigen in a concentration gradient manner, and was detected with an Ab generated against the mouse CD40L at all concentrations Fig 5 .",
"Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence, an Ab known to detect mouse CD40L was used to identify the purified crCD40L protein. The purified recombinant crCD40L was used as a coating antigen in a concentration gradient manner, and was detected with an Ab generated against the mouse CD40L at all concentrations Fig 5 . Uncoated controls were performed in parallel and were negative for CD40L in ELISA. We measured the overall strength of the antigen-antibody complex in the presence of 6M urea . The avidity of the cotton rat CD40L for the anti-mouse CD40L antibody was decreased in the presence of 6M urea at all concentrations. Clearly, as the antibody used was raised against mouse CD40L, the crCD40L is detected by mouse CD40L.",
"The avidity of the cotton rat CD40L for the anti-mouse CD40L antibody was decreased in the presence of 6M urea at all concentrations. Clearly, as the antibody used was raised against mouse CD40L, the crCD40L is detected by mouse CD40L. crCD40L was expressed in vaccinia virus and purified from infected BHK21 cell lysate on a nickel column. The purified protein was detected by ELISA using a mouse antibody against CD40L in a concentration gradient dependent manner. The avidity of the mouse CD40L antibody to the cotton rat CD40L protein was evaluated in the presence of 6M urea. The difference between the untreated and 6M urea treated for each group was calculated using students t-test ÃÃÃ p<0.001, ÃÃÃÃ p<0.0001 n = 2 .",
"The avidity of the mouse CD40L antibody to the cotton rat CD40L protein was evaluated in the presence of 6M urea. The difference between the untreated and 6M urea treated for each group was calculated using students t-test ÃÃÃ p<0.001, ÃÃÃÃ p<0.0001 n = 2 . Data shown is a representative experiment of three separate experiments where two n = 2 technical replicates are conducted in each experiment. The no-coating and noprimary antibody negative controls gave average OD values of 0.56 and 0.107 respectively. addition of urea treatment would substantially weaken the interaction between the antibody and crCD40L. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence with similar functional domains, we evaluated the biological activity of the recombinant crCD40L on immature murine bone marrow DCs.",
"addition of urea treatment would substantially weaken the interaction between the antibody and crCD40L. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence with similar functional domains, we evaluated the biological activity of the recombinant crCD40L on immature murine bone marrow DCs. We conducted experiments based on known functional activities of CD40L in other animal species. Specifically, maturation of immature DCs after exposure to antigen is known to play a crucial role in their immunity-stimulating function , while trimeric recombinant CD40L has been shown to stimulate DC immunomodulating functions . When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated .",
"When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L . Moreover, mouse I-A d major histocompatibility complex is also up-regulated upon stimulation with CD40L . When our recombinant crCD40L was used to stimulate immature murine bone marrow DCs, we observed similar results to that when murine CD40L is used Tables 1 and 2 . CD11c was down regulated in both median flouresence intensity Table 1 and the percentage of positive cells Table 2 .",
"When our recombinant crCD40L was used to stimulate immature murine bone marrow DCs, we observed similar results to that when murine CD40L is used Tables 1 and 2 . CD11c was down regulated in both median flouresence intensity Table 1 and the percentage of positive cells Table 2 . The co-stimulatory molecules CD54, CD40, CD80, and CD86 were all up-regulated in both median fluorescence intensity Table 1 and the percentage of positive cells Table 2 . The Mouse I-A d major histocompatibility complex was upregulated in median fluorescence intensity Table 1 but not up-regulated in terms of the overall percentage of positive cells Table 2 . We speculate this to be due to the species incompatibility since we are stimulating mouse bone marrow cells with cotton rat CD40L. Nevertheless, the crCD40L was able to promote up-regulation of key co-stimulatory markers on immature DCs promoting DC maturation.",
"We speculate this to be due to the species incompatibility since we are stimulating mouse bone marrow cells with cotton rat CD40L. Nevertheless, the crCD40L was able to promote up-regulation of key co-stimulatory markers on immature DCs promoting DC maturation. The gating strategy used for the flow cytometry analysis is provided in S3 Fig along with overlapping histograms of the intracellular adhesion marker and co-stimulatory markers. CD40-induced activation of cytokine gene expression in DCs by CD40L is an important process in the initiation of primary immune responses and is critical for DC maturation and the generation of antigen-specific T cell responses . IL-6 is a highly pleiotropic cytokine in that it stimulates the activation, proliferation, and survival of T cells, and furthermore, modifies DC function and survival . We tested if the recombinant crCD40L could induce IL-6 gene expression Fig 6a and production of the cytokine Fig 6b by immature murine bone marrow DCs.",
"IL-6 is a highly pleiotropic cytokine in that it stimulates the activation, proliferation, and survival of T cells, and furthermore, modifies DC function and survival . We tested if the recombinant crCD40L could induce IL-6 gene expression Fig 6a and production of the cytokine Fig 6b by immature murine bone marrow DCs. The results indicate that a significant increase in both IL-6 gene expression and cytokine production in immature murine bone marrow DCs was observed forty hours after stimulation with the crCD40L. Collectively, the observation that both the upregulation of immature DC cell surface maturation markers and increased IL-6 gene expression and cytokine production provide strong evidence of the biological activity of crCD40L. In summary, the cotton rat CD40L cDNA that we isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783 bp which coded for a 260 aa protein. The recombinant cotton rat CD40L was recognized by an Ab against mouse CD40L in direct ELISA, and showed biological activity by upregulating maturation markers CD40, CD54, CD80, and CD86 as well as I-A d on immature bone marrow murine DCs and moreover, inducing upregulation of IL-6 gene and cytokine expression in these cells.",
"In summary, the cotton rat CD40L cDNA that we isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783 bp which coded for a 260 aa protein. The recombinant cotton rat CD40L was recognized by an Ab against mouse CD40L in direct ELISA, and showed biological activity by upregulating maturation markers CD40, CD54, CD80, and CD86 as well as I-A d on immature bone marrow murine DCs and moreover, inducing upregulation of IL-6 gene and cytokine expression in these cells. The isolation of the cotton rat CD40L sequence and availability of CD40L has the potential to positively impact basic immunological research and vaccine development, given the critical importance of this protein in orchestrating immune responses ."
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What is the effect of CD40L on Dendritic Cells?
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it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells
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[
"Cotton rats are an important animal model to study infectious diseases. They have demonstrated higher susceptibility to a wider variety of human pathogens than other rodents and are also the animal model of choice for pre-clinical evaluations of some vaccine candidates. However, the genome of cotton rats remains to be fully sequenced, with much fewer genes cloned and characterised compared to other rodent species. Here we report the cloning and characterization of CD40 ligand, whose human and murine counterparts are known to be expressed on a range of cell types including activated T cells and B cells, dendritic cells, granulocytes, macrophages and platelets and exerts a broad array of immune responses. The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame ORF of 783bp coding for a 260 amino acid protein. The recombinant cotton rat CD40L protein was recognized by an antibody against mouse CD40L.",
"The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame ORF of 783bp coding for a 260 amino acid protein. The recombinant cotton rat CD40L protein was recognized by an antibody against mouse CD40L. Moreover, it demonstrated functional activities on immature bone marrow dendritic cells by upregulating surface maturation markers CD40, CD54, CD80, and CD86 , and increasing IL-6 gene and protein expression. The availability of CD40L gene identity could greatly facilitate mechanistic research on pathogen-induced-immunopathogenesis and vaccine-elicited immune responses. Text: The cotton rat Sigmodon hispidus was first used in polio research in the 1930s , and throughout the last century, it has proven to be an excellent model for biomedical research . Historically in biomedical research, the mouse has been exploited as the default animal model.",
"Text: The cotton rat Sigmodon hispidus was first used in polio research in the 1930s , and throughout the last century, it has proven to be an excellent model for biomedical research . Historically in biomedical research, the mouse has been exploited as the default animal model. This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus . This model has been valuable for adenovirus-based gene replacement therapy research , and was also proven to be indispensable in pre-clinical evaluation of the prophylactic antibodies RespiGam 1 , and Synagis 1 .",
"On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus . This model has been valuable for adenovirus-based gene replacement therapy research , and was also proven to be indispensable in pre-clinical evaluation of the prophylactic antibodies RespiGam 1 , and Synagis 1 . Indeed, the cotton rat model was found to be valuable in terms of its biological and immunological relevance, it was deemed unnecessary to test the adenovirus-based gene therapy and the Synagis 1 prophylactic treatment against RSV disease in non-human primate prior to the human trials . A number of methods and reagents have been developed for the analysis of immune responses in cotton rats over the last decade. Up to date, more than 200 genes encoding cytokines, chemokines, cell surface markers and regulatory molecules have been cloned, with various related research reagents being commercially available. As a result, the use of cotton rats in pathogenesis studies addressing mechanistic questions has significantly increased.",
"Up to date, more than 200 genes encoding cytokines, chemokines, cell surface markers and regulatory molecules have been cloned, with various related research reagents being commercially available. As a result, the use of cotton rats in pathogenesis studies addressing mechanistic questions has significantly increased. Nevertheless, the gene encoding CD154 and CD40 ligand CD40L , remains elusive. CD40L plays a critical role in orchestrating immune responses against pathogens. Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands .",
"Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets . It has been shown that upon interacting with its receptor, CD40, CD40L induces profound effects on T cells, DCs, B cells, endothelial cells, as well as many cells of the hematopoietic and non-hematopoietic systems. Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin Ig isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy.",
"Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin Ig isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy. It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes . Here we report the successful cloning of the gene encoding cotton rat CD40L crCD40L ; we also expressed and purified the CD40L produced in mammalian cells. Further characterisation of the recombinant cotton rat CD40L revealed its functional activities in promoting DC maturation and cytokine production. weeks old cotton rats were obtained from an inbred colony maintained at Envigo USA .",
"Further characterisation of the recombinant cotton rat CD40L revealed its functional activities in promoting DC maturation and cytokine production. weeks old cotton rats were obtained from an inbred colony maintained at Envigo USA . All animal experiments were conducted in accordance with Institutional Care and Use Committee IACUC of Health Canada Ottawa Animal Care Committee which approved this study. The rats were housed 3 animals per cage in Allentown NexGen individually ventilated cages with free access to food and water. These cages provided a floor space of 142 in 2 / 916 cm 2 . Body weight and any sign of distress were monitored daily. If anything associated the animal health was observed, a full examination would be conducted.",
"Body weight and any sign of distress were monitored daily. If anything associated the animal health was observed, a full examination would be conducted. As In this study spleen cells from normal, healthy animals were isolated, we did not observe any adverse reaction. To isolate splenocytes from the animals, isoflourane was used to put the animals to sleep via inhalation with oxygen for euthanasia. The spleens from three naïve cotton rats were removed aseptically and snap frozen in liquid nitrogen. The spleens were homogenized individually with a TissueLyser II Qiagen and total RNA extracted using the RNeasy Mini kit Qiagen with on-column DNase digestion according to the user's manual.",
"The spleens from three naïve cotton rats were removed aseptically and snap frozen in liquid nitrogen. The spleens were homogenized individually with a TissueLyser II Qiagen and total RNA extracted using the RNeasy Mini kit Qiagen with on-column DNase digestion according to the user's manual. The 3' RACE system Life Technologies was then used with to amplify the 3' portion of the cotton rat CD40L from the total RNA according to the manufacturer's instructions. A schematic of the 3' RACE procedure used is provided in S1 Fig. A gene specific primer 5'-GGACTCTATTATGTCTACACCCAAGTCACCTTCTG -3' was derived from a consensus sequence aligning the rat Rattus norvegicus UniProt: Q9Z2V2 , mouse Mus musculus UniProt: P27548 , and golden hamster Mesocricetus auratus NCBI Reference Sequence: XM_005084522.3 CD40L sequences obtained from the National Center for Biotechnology Information NCBI . Following first strand cDNA synthesis, the 3' portion of the cotton rat CD40L mRNA was PCR amplified using the consensus sequence derived gene specific primer and the abridged universal amplification primer with an annealing temperature at 56˚C.",
"A gene specific primer 5'-GGACTCTATTATGTCTACACCCAAGTCACCTTCTG -3' was derived from a consensus sequence aligning the rat Rattus norvegicus UniProt: Q9Z2V2 , mouse Mus musculus UniProt: P27548 , and golden hamster Mesocricetus auratus NCBI Reference Sequence: XM_005084522.3 CD40L sequences obtained from the National Center for Biotechnology Information NCBI . Following first strand cDNA synthesis, the 3' portion of the cotton rat CD40L mRNA was PCR amplified using the consensus sequence derived gene specific primer and the abridged universal amplification primer with an annealing temperature at 56˚C. The reverse complementary sequence of this primer was then used as a reverse primer with the forward primer 5'-GATAGAAACATACAGCCAACCTTCTCCCAGATC -3' to amplify the 5' portion of the cotton rat CD40L mRNA with an annealing temperature of 57˚C. All amplified fragments were sequenced with BigDye Terminator v.3.1 Cycle Sequencing kit ThermoFisher cat # 4336917 . Briefly, samples were amplified in a PTC-200 thermal cycle MJ Research with the following program: 26 cycles of 1˚C/S to 96˚C, 96˚C for 10 seconds, 1˚C/S to 50˚C, 50˚C for 5 seconds, 1˚C/S to 60˚C, 60˚C for 4 minutes. The samples were cleaned using DyeEx 2.0 Spin kit Qiagen cat # 63204 and loaded onto a 3130xl Genetic Analyzer Applied Biosystems .",
"Briefly, samples were amplified in a PTC-200 thermal cycle MJ Research with the following program: 26 cycles of 1˚C/S to 96˚C, 96˚C for 10 seconds, 1˚C/S to 50˚C, 50˚C for 5 seconds, 1˚C/S to 60˚C, 60˚C for 4 minutes. The samples were cleaned using DyeEx 2.0 Spin kit Qiagen cat # 63204 and loaded onto a 3130xl Genetic Analyzer Applied Biosystems . Raw sequencing data was edited by the instrument's software ThermoFisher 3130xl Genetic Analyzer Data Collection Software v3.0 , and then imported into GeneCodes Sequencher v4.6.1 sequencing analysis software for further editing. The final sequenced contigs are then imported to NCBI BLAST cgi to confirm the identity. Putative conserved domains, trimer interface, and receptor binding sites were determined by performing a standard protein BLAST blastp algorithm; The sequences producing significant alignments were imported into Geneosis software, Auckland, New Zealand . Multiple alignment was conducted as previously described , with phylogenetic analysis using Geneosis Pro 5.6.7.",
"Putative conserved domains, trimer interface, and receptor binding sites were determined by performing a standard protein BLAST blastp algorithm; The sequences producing significant alignments were imported into Geneosis software, Auckland, New Zealand . Multiple alignment was conducted as previously described , with phylogenetic analysis using Geneosis Pro 5.6.7. Once the mRNA sequence was confirmed, a construct was designed beginning with a kozak sequence 5'-CACCGCCGCCACC-3' , followed by a secretion signal consisting of 23 amino acid aa MLLAVLYCLLWSFQTSAGHFPRA from the human tyrosinase signal peptide as previously described . This is followed by six histidine residues to facilitate protein purification. Following this sequence, a 27-aa fragment from the bacteriophage T4 fibritin trimerization motif was added and finally connected to the full length 783bp open reading frame ORF of the cotton rat CD40L sequence at the C terminus. This construct was synthesized and cloned into pUC57 Biobasic, Markham, ON .",
"Following this sequence, a 27-aa fragment from the bacteriophage T4 fibritin trimerization motif was added and finally connected to the full length 783bp open reading frame ORF of the cotton rat CD40L sequence at the C terminus. This construct was synthesized and cloned into pUC57 Biobasic, Markham, ON . Generation of a recombinant vaccinia virus expressing cotton rat CD40L protein construct was achieved using a vaccinia virus E3L and K3L double deletion mutant virus as the parental virus and taterapoxvirus K3L as the positive selection marker Jingxin Cao, unpublished information . Briefly, the recombination plasmid vector for expression of the CD40L construct gene consists of the homologous flanking vaccinia DNA sequences targeting vaccinia A45R gene SOD homolog ; the CD40L construct gene driven by a modified vaccinia H5 promoter Vaccine 1996, 14:1451 , and taterapoxvirus 037 gene driven by vaccinia K3L promoter as the positive selection marker. The recombination vector was transfected into a HeLa PKR knockout cells infected with a vaccinia virus with both E3L and K3L genes deleted. Selection and purification of the recombinant vaccinia virus expressing the CD40L was done in BHK21 cells.",
"The recombination vector was transfected into a HeLa PKR knockout cells infected with a vaccinia virus with both E3L and K3L genes deleted. Selection and purification of the recombinant vaccinia virus expressing the CD40L was done in BHK21 cells. Expression of the CD40L protein was confirmed by Western blotting using His-tag Ab. Cell monolayers were lysed in sample buffer and homogenized using QIAshredder columns Qiagen . Western blotting was performed using 4 to 15% TGX gel and Tris/Glycine/SDS running buffer Bio-Rad Laboratories Inc. , and the protein samples were transferred to Immobilon-FL PVDF membranes Millipore . Protein was detected with Tetra-HIS Ab Qiagen and goat anti-mouse IRDye-800CW LiCor . Membranes were developed using the Odyssey system LiCor .",
"Protein was detected with Tetra-HIS Ab Qiagen and goat anti-mouse IRDye-800CW LiCor . Membranes were developed using the Odyssey system LiCor . The vaccinia virus carrying the crCD40L gene was propagated in BHK21 cells. The cells were collected and washed with PBS once and then lysed with a denaturing buffer 10 mM Tris-HCl, 100 mM sodium phosphate, 6 M guanidine hydrochloride, 10 mM reduced glutathione, pH 8.0 and disrupted by sonication on ice using a Branson sonifier 150 ThermoFisher, Waltham, MA at level 1 for two 10sec bursts with 1min rest on ice between. After separation of cell debris, the supernatant was added to a slurry of Ni-NTA resin Qiagen, Mississauga, ON, Canada 10 mL resin bed and stirred at room temperature for 30 min before loading into a column. The column was purified using an AKTA purifier Amersham Biosciences with Unicorn 5.3 software Amersham Biosciences .",
"After separation of cell debris, the supernatant was added to a slurry of Ni-NTA resin Qiagen, Mississauga, ON, Canada 10 mL resin bed and stirred at room temperature for 30 min before loading into a column. The column was purified using an AKTA purifier Amersham Biosciences with Unicorn 5.3 software Amersham Biosciences . Refolding was accomplished under oxidative conditions with a gradient of denaturing buffer to buffer B buffer B: 10 mM Tris-HCl, 100 mM sodium phosphate, pH 7.8 over 10 column volumes CVs . The column was then washed with three CVs of buffer B + 60 mM imidazole pH 7.8 to remove unspecific binding. The protein was eluted off the column with buffer B + 250 mM imidazole pH 7.8 . The resulting protein was dialysed against PBS pH 7.5 and then confirmed by western blot.",
"The protein was eluted off the column with buffer B + 250 mM imidazole pH 7.8 . The resulting protein was dialysed against PBS pH 7.5 and then confirmed by western blot. 96-well plates were coated with either recombinant mouse CD40L R&D Systems or the recombinant crCD40L protein 2ug/ml in 100μl PBS. Plates were washed with wash buffer PBS-0.1% tween-20 and then blocked with 200μl/well blocking buffer PBS containing 0.1% Tween 20 and 3%IgG Free BSA for 1 hour at 37˚C. Plates were washed with wash buffer and incubated at 37˚C for 1 hour with 100μl/well goat anti-mouseCD40L R&D Systems 2ug/ml in blocking buffer. Plates were subsequently washed and incubated at 37˚C for 1 hour with 100μl/well with rabbit anti-goat IgG HRP conjugate Zymed .",
"Plates were washed with wash buffer and incubated at 37˚C for 1 hour with 100μl/well goat anti-mouseCD40L R&D Systems 2ug/ml in blocking buffer. Plates were subsequently washed and incubated at 37˚C for 1 hour with 100μl/well with rabbit anti-goat IgG HRP conjugate Zymed . Plates were washed again and incubated for 10 min in the dark with 100μl/well 3,3'5,5'-tetramethylbenzidine substrate New England Bio Labs . The reaction was stopped with Stop solution New England Bio Labs and absorbance was read at 450nm on a BioTek Synergy 2 plate reader. Primary bone marrow cells from Balb/c mice Chicago, IL were thawed and cultured in dendritic cell medium from manufacture Cell Biologics M7711 supplemented with GMCSF Cell Biologics without IL-4 at 4x10 5 cells/well in a volume of 200μl. The cells were treated with 0.5μg/ml recombinant mouse CD40L Preprotech, Montreal, QC or the recombinant crCD40L protein at 0.5μg/ml, 5μg/ml, or 50μg/ml.",
"Primary bone marrow cells from Balb/c mice Chicago, IL were thawed and cultured in dendritic cell medium from manufacture Cell Biologics M7711 supplemented with GMCSF Cell Biologics without IL-4 at 4x10 5 cells/well in a volume of 200μl. The cells were treated with 0.5μg/ml recombinant mouse CD40L Preprotech, Montreal, QC or the recombinant crCD40L protein at 0.5μg/ml, 5μg/ml, or 50μg/ml. Forty hours later, flow cytometry was performed on a BD LSRFortessa cell analyser after 2 x 10 5 cells/tube were stained using CD11c-PE-CF594, CD54-FITC, CD40-BV786, CD80-BV421, and CD86-BV711 antibodies. All antibodies were purchased from BD Biosciences. The resulting spectra were analysed using FACS-Diva version 8.0.1 software. To assess IL-6 mRNA production of immature bone marrow murine DCs in response to targeting by recombinant crCD40L, quantitative real-time PCR was conducted on an ABI Prism 7500 Fast Sequence detection system Applied Biosystems .",
"The resulting spectra were analysed using FACS-Diva version 8.0.1 software. To assess IL-6 mRNA production of immature bone marrow murine DCs in response to targeting by recombinant crCD40L, quantitative real-time PCR was conducted on an ABI Prism 7500 Fast Sequence detection system Applied Biosystems . TaqMan assay reagent kits Applied Biosystems were used that contain pre-standardized primers and TaqMan MGB probes for IL-6 and 18S which were used to normalize the data. Total RNA was isolated from 8x10 5 stimulated bone marrow DCs using the RNeasy Mini Kit Qiagen according to manufactures instructions. The isolated RNA was used to make cDNA using the Superscript III First-Strand Synthesis System for RT-PCR Invitrogen according to manufacturer's instructions. The cDNA was then subjected to quantitative PCR using the TaqMan Fast Advanced Master Mix Applied Biosystems according to manufactures instructions.",
"The isolated RNA was used to make cDNA using the Superscript III First-Strand Synthesis System for RT-PCR Invitrogen according to manufacturer's instructions. The cDNA was then subjected to quantitative PCR using the TaqMan Fast Advanced Master Mix Applied Biosystems according to manufactures instructions. Samples were run in duplicate and C t values were obtained. Fold change over unstimulated DCs was calculated using the 2 -ΔΔCT method of relative quantification , using 18S as the housekeeping reference gene. To investigate IL-6 secretion by murine bone marrow DCs, supernatant from forty hour stimulated cultures were collected and assayed using the Mouse IL-6 DuoSet ELISA Kit R & D Systems following the manufacturer's protocol. The complete mRNA sequence of CD40L was obtained in two steps Fig 1 .",
"To investigate IL-6 secretion by murine bone marrow DCs, supernatant from forty hour stimulated cultures were collected and assayed using the Mouse IL-6 DuoSet ELISA Kit R & D Systems following the manufacturer's protocol. The complete mRNA sequence of CD40L was obtained in two steps Fig 1 . A sequence corresponding to nucleotides 535 through to the poly-A tail was obtained using the 3' RACE kit and mRNA as starting material, which was isolated from cotton rat splenocytes and a rodent consensus sequence as a primer. This portion of the sequence has the 3' un-translated region of the mRNA as well as the stop codon. The 5' end of the protein was obtained in the next step by PCR amplification of the cDNA obtained in the first step with the 3' RACE kit and the reverse complement of the consensus sequence primer and a second consensus sequence primer designed to bind to the beginning of the CD40L mRNA. The 783bp ORF encodes 260aa followed by a stop codon.",
"The 5' end of the protein was obtained in the next step by PCR amplification of the cDNA obtained in the first step with the 3' RACE kit and the reverse complement of the consensus sequence primer and a second consensus sequence primer designed to bind to the beginning of the CD40L mRNA. The 783bp ORF encodes 260aa followed by a stop codon. Comparison of the sequenced CD40L gene revealed that the crCD40L coding sequence shares 93%, 89%, and 83%, identity with golden hamster, rat, and mouse, respectively. At the amino acid aa level, the corresponding identities are 91%, 82%, and 82%, Fig 2a. At both the mRNA and aa levels, the crCD40L shared the closest similarity with Peromyscus maniculatus bairdii or deer mouse at 93% and 92% respectively. When sequence homology analysis is performed, crCD40L clusters with other members of the Cricetidae family Fig 2b.",
"At both the mRNA and aa levels, the crCD40L shared the closest similarity with Peromyscus maniculatus bairdii or deer mouse at 93% and 92% respectively. When sequence homology analysis is performed, crCD40L clusters with other members of the Cricetidae family Fig 2b. We next examined the functional domains in crCD40L in comparison with other known CD40L. As shown in Fig 3a, crCD40L has a putative tumor necrosis factor TNF superfamily Using EZmol software , we predicted folding of the protein as shown in Fig 3b. The cotton rat CD40L cDNA that we have isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783bp which coded for a 260 aa protein. The homology of cotton rat CD40L, at both the amino acid and nucleic acid level, is closer to members of the Cricetidae family hamster and deer mouse than to those of the Muridae family rat and mouse as shown in Fig 2b.",
"The cotton rat CD40L cDNA that we have isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783bp which coded for a 260 aa protein. The homology of cotton rat CD40L, at both the amino acid and nucleic acid level, is closer to members of the Cricetidae family hamster and deer mouse than to those of the Muridae family rat and mouse as shown in Fig 2b. As with other known CD40L proteins, there is a putative TNF superfamily domain, a transmembrane domain, trimerization sites, and receptor binding sites . TNF superfamily members include TNF TNF-alpha , LT lymphotoxin-alpha, TNF-beta , CD40 ligand, Apo2L TRAIL , Fas ligand, and osteoprotegerin OPG ligand, among others . The TNF superfamily is composed of 19 ligands and 29 receptors, in which each has vastly diversified roles in the body and exhibit pro-inflammatory activity, partly via activation of NF-kB . Members of this family generally have an intracellular N-terminal domain, a short transmembrane segment, an extracellular stalk, and a globular TNF-like extracellular domain of about 150 residues .",
"The TNF superfamily is composed of 19 ligands and 29 receptors, in which each has vastly diversified roles in the body and exhibit pro-inflammatory activity, partly via activation of NF-kB . Members of this family generally have an intracellular N-terminal domain, a short transmembrane segment, an extracellular stalk, and a globular TNF-like extracellular domain of about 150 residues . They initiate apoptosis by binding to related receptors, some of which have intracellular death domains . These proteins typically form homo-or hetero-trimeric complexes and bind one elongated receptor molecule along each of three clefts formed by neighboring monomers of the trimer and ligand trimerization is for receptor binding . All seven known conserved residues that constitute the trimer interface on the conserved TNF domain , were mapped to the putative crCD40L protein sequence. Additionally, all six known conserved receptor binding sites on the conserved TNF domain , were mapped to the crCD40L protein sequence.",
"All seven known conserved residues that constitute the trimer interface on the conserved TNF domain , were mapped to the putative crCD40L protein sequence. Additionally, all six known conserved receptor binding sites on the conserved TNF domain , were mapped to the crCD40L protein sequence. In order to further evaluate the crCD40L deduced sequence, the full 783bp ORF of the crCD40L was cloned into a vaccinia virus vector. The crCD40L construct was designed to carry a secretion signal, histidine tag, and a trimerization motif Fig 4a . Selection and purification of the recombinant vaccinia virus expressing the CD40L construct was conducted in BHK21 cells. Western blot with anti-histidine antibody Ab was used to confirm expression of the CD40L protein construct Fig 4b and S2 Fig.",
"Selection and purification of the recombinant vaccinia virus expressing the CD40L construct was conducted in BHK21 cells. Western blot with anti-histidine antibody Ab was used to confirm expression of the CD40L protein construct Fig 4b and S2 Fig. The resulting 36 kDa protein product was found in both the cell lysate and supernatant faint band-48 hours only . Since the highest expression was found in the cell lysate, it was used for further purification of the protein. It should be noted that the protein was only able to be detected under reducing conditions. Under non-reducing conditions, the protein was unable to be detected by the anti-histidine Ab, even in the cell lysate data not shown .",
"It should be noted that the protein was only able to be detected under reducing conditions. Under non-reducing conditions, the protein was unable to be detected by the anti-histidine Ab, even in the cell lysate data not shown . This indicates that the histidine tag is folded within the trimer and is unavailable in the native form for purification. This is an additional reason for the need to purify the protein from the cell lysate under harsh denaturing conditions followed by protein refolding. The reason we utilized a mammalian expression system to produce the protein rather than a bacterial system is to facilitate its proper folding into its native structure, trimerization, and glycosylation. The aa backbone predicts a protein of 29 kDa, yet initial studies of the CD40L protein suggested a molecular mass of 39 kDa, and on most cell types the molecular mass of CD40L is 32-33kDa, consistent with extensive post-translation modification .",
"The reason we utilized a mammalian expression system to produce the protein rather than a bacterial system is to facilitate its proper folding into its native structure, trimerization, and glycosylation. The aa backbone predicts a protein of 29 kDa, yet initial studies of the CD40L protein suggested a molecular mass of 39 kDa, and on most cell types the molecular mass of CD40L is 32-33kDa, consistent with extensive post-translation modification . The BHK21 cells expressing the crCD40L construct were collected and lysed with 6 M guanidine hydrochloride with reduced glutathione and sonication. The lysate was loaded on the nickel column and the washed with denaturing buffer as described in materials and methods. The bound proteins were refolded on the column with gradient buffer exchange, to allow slow refold the protein, given that CD40L biological activity is dependent on a homo-trimer configuration . The resulting bound protein was subsequently eluted with imidazole.",
"The bound proteins were refolded on the column with gradient buffer exchange, to allow slow refold the protein, given that CD40L biological activity is dependent on a homo-trimer configuration . The resulting bound protein was subsequently eluted with imidazole. The resulting fractions that showed a peak were pooled and dialysed against PBS. The purified protein was confirmed in ELISA. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence, an Ab known to detect mouse CD40L was used to identify the purified crCD40L protein. The purified recombinant crCD40L was used as a coating antigen in a concentration gradient manner, and was detected with an Ab generated against the mouse CD40L at all concentrations Fig 5 .",
"Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence, an Ab known to detect mouse CD40L was used to identify the purified crCD40L protein. The purified recombinant crCD40L was used as a coating antigen in a concentration gradient manner, and was detected with an Ab generated against the mouse CD40L at all concentrations Fig 5 . Uncoated controls were performed in parallel and were negative for CD40L in ELISA. We measured the overall strength of the antigen-antibody complex in the presence of 6M urea . The avidity of the cotton rat CD40L for the anti-mouse CD40L antibody was decreased in the presence of 6M urea at all concentrations. Clearly, as the antibody used was raised against mouse CD40L, the crCD40L is detected by mouse CD40L.",
"The avidity of the cotton rat CD40L for the anti-mouse CD40L antibody was decreased in the presence of 6M urea at all concentrations. Clearly, as the antibody used was raised against mouse CD40L, the crCD40L is detected by mouse CD40L. crCD40L was expressed in vaccinia virus and purified from infected BHK21 cell lysate on a nickel column. The purified protein was detected by ELISA using a mouse antibody against CD40L in a concentration gradient dependent manner. The avidity of the mouse CD40L antibody to the cotton rat CD40L protein was evaluated in the presence of 6M urea. The difference between the untreated and 6M urea treated for each group was calculated using students t-test ÃÃÃ p<0.001, ÃÃÃÃ p<0.0001 n = 2 .",
"The avidity of the mouse CD40L antibody to the cotton rat CD40L protein was evaluated in the presence of 6M urea. The difference between the untreated and 6M urea treated for each group was calculated using students t-test ÃÃÃ p<0.001, ÃÃÃÃ p<0.0001 n = 2 . Data shown is a representative experiment of three separate experiments where two n = 2 technical replicates are conducted in each experiment. The no-coating and noprimary antibody negative controls gave average OD values of 0.56 and 0.107 respectively. addition of urea treatment would substantially weaken the interaction between the antibody and crCD40L. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence with similar functional domains, we evaluated the biological activity of the recombinant crCD40L on immature murine bone marrow DCs.",
"addition of urea treatment would substantially weaken the interaction between the antibody and crCD40L. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence with similar functional domains, we evaluated the biological activity of the recombinant crCD40L on immature murine bone marrow DCs. We conducted experiments based on known functional activities of CD40L in other animal species. Specifically, maturation of immature DCs after exposure to antigen is known to play a crucial role in their immunity-stimulating function , while trimeric recombinant CD40L has been shown to stimulate DC immunomodulating functions . When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated .",
"When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L . Moreover, mouse I-A d major histocompatibility complex is also up-regulated upon stimulation with CD40L . When our recombinant crCD40L was used to stimulate immature murine bone marrow DCs, we observed similar results to that when murine CD40L is used Tables 1 and 2 . CD11c was down regulated in both median flouresence intensity Table 1 and the percentage of positive cells Table 2 .",
"When our recombinant crCD40L was used to stimulate immature murine bone marrow DCs, we observed similar results to that when murine CD40L is used Tables 1 and 2 . CD11c was down regulated in both median flouresence intensity Table 1 and the percentage of positive cells Table 2 . The co-stimulatory molecules CD54, CD40, CD80, and CD86 were all up-regulated in both median fluorescence intensity Table 1 and the percentage of positive cells Table 2 . The Mouse I-A d major histocompatibility complex was upregulated in median fluorescence intensity Table 1 but not up-regulated in terms of the overall percentage of positive cells Table 2 . We speculate this to be due to the species incompatibility since we are stimulating mouse bone marrow cells with cotton rat CD40L. Nevertheless, the crCD40L was able to promote up-regulation of key co-stimulatory markers on immature DCs promoting DC maturation.",
"We speculate this to be due to the species incompatibility since we are stimulating mouse bone marrow cells with cotton rat CD40L. Nevertheless, the crCD40L was able to promote up-regulation of key co-stimulatory markers on immature DCs promoting DC maturation. The gating strategy used for the flow cytometry analysis is provided in S3 Fig along with overlapping histograms of the intracellular adhesion marker and co-stimulatory markers. CD40-induced activation of cytokine gene expression in DCs by CD40L is an important process in the initiation of primary immune responses and is critical for DC maturation and the generation of antigen-specific T cell responses . IL-6 is a highly pleiotropic cytokine in that it stimulates the activation, proliferation, and survival of T cells, and furthermore, modifies DC function and survival . We tested if the recombinant crCD40L could induce IL-6 gene expression Fig 6a and production of the cytokine Fig 6b by immature murine bone marrow DCs.",
"IL-6 is a highly pleiotropic cytokine in that it stimulates the activation, proliferation, and survival of T cells, and furthermore, modifies DC function and survival . We tested if the recombinant crCD40L could induce IL-6 gene expression Fig 6a and production of the cytokine Fig 6b by immature murine bone marrow DCs. The results indicate that a significant increase in both IL-6 gene expression and cytokine production in immature murine bone marrow DCs was observed forty hours after stimulation with the crCD40L. Collectively, the observation that both the upregulation of immature DC cell surface maturation markers and increased IL-6 gene expression and cytokine production provide strong evidence of the biological activity of crCD40L. In summary, the cotton rat CD40L cDNA that we isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783 bp which coded for a 260 aa protein. The recombinant cotton rat CD40L was recognized by an Ab against mouse CD40L in direct ELISA, and showed biological activity by upregulating maturation markers CD40, CD54, CD80, and CD86 as well as I-A d on immature bone marrow murine DCs and moreover, inducing upregulation of IL-6 gene and cytokine expression in these cells.",
"In summary, the cotton rat CD40L cDNA that we isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783 bp which coded for a 260 aa protein. The recombinant cotton rat CD40L was recognized by an Ab against mouse CD40L in direct ELISA, and showed biological activity by upregulating maturation markers CD40, CD54, CD80, and CD86 as well as I-A d on immature bone marrow murine DCs and moreover, inducing upregulation of IL-6 gene and cytokine expression in these cells. The isolation of the cotton rat CD40L sequence and availability of CD40L has the potential to positively impact basic immunological research and vaccine development, given the critical importance of this protein in orchestrating immune responses ."
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What is the effect of CD40L on B Cells?
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it promotes germinal center formation, immunoglobulin (Ig) isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells
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"Cotton rats are an important animal model to study infectious diseases. They have demonstrated higher susceptibility to a wider variety of human pathogens than other rodents and are also the animal model of choice for pre-clinical evaluations of some vaccine candidates. However, the genome of cotton rats remains to be fully sequenced, with much fewer genes cloned and characterised compared to other rodent species. Here we report the cloning and characterization of CD40 ligand, whose human and murine counterparts are known to be expressed on a range of cell types including activated T cells and B cells, dendritic cells, granulocytes, macrophages and platelets and exerts a broad array of immune responses. The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame ORF of 783bp coding for a 260 amino acid protein. The recombinant cotton rat CD40L protein was recognized by an antibody against mouse CD40L.",
"The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame ORF of 783bp coding for a 260 amino acid protein. The recombinant cotton rat CD40L protein was recognized by an antibody against mouse CD40L. Moreover, it demonstrated functional activities on immature bone marrow dendritic cells by upregulating surface maturation markers CD40, CD54, CD80, and CD86 , and increasing IL-6 gene and protein expression. The availability of CD40L gene identity could greatly facilitate mechanistic research on pathogen-induced-immunopathogenesis and vaccine-elicited immune responses. Text: The cotton rat Sigmodon hispidus was first used in polio research in the 1930s , and throughout the last century, it has proven to be an excellent model for biomedical research . Historically in biomedical research, the mouse has been exploited as the default animal model.",
"Text: The cotton rat Sigmodon hispidus was first used in polio research in the 1930s , and throughout the last century, it has proven to be an excellent model for biomedical research . Historically in biomedical research, the mouse has been exploited as the default animal model. This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus . This model has been valuable for adenovirus-based gene replacement therapy research , and was also proven to be indispensable in pre-clinical evaluation of the prophylactic antibodies RespiGam 1 , and Synagis 1 .",
"On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus . This model has been valuable for adenovirus-based gene replacement therapy research , and was also proven to be indispensable in pre-clinical evaluation of the prophylactic antibodies RespiGam 1 , and Synagis 1 . Indeed, the cotton rat model was found to be valuable in terms of its biological and immunological relevance, it was deemed unnecessary to test the adenovirus-based gene therapy and the Synagis 1 prophylactic treatment against RSV disease in non-human primate prior to the human trials . A number of methods and reagents have been developed for the analysis of immune responses in cotton rats over the last decade. Up to date, more than 200 genes encoding cytokines, chemokines, cell surface markers and regulatory molecules have been cloned, with various related research reagents being commercially available. As a result, the use of cotton rats in pathogenesis studies addressing mechanistic questions has significantly increased.",
"Up to date, more than 200 genes encoding cytokines, chemokines, cell surface markers and regulatory molecules have been cloned, with various related research reagents being commercially available. As a result, the use of cotton rats in pathogenesis studies addressing mechanistic questions has significantly increased. Nevertheless, the gene encoding CD154 and CD40 ligand CD40L , remains elusive. CD40L plays a critical role in orchestrating immune responses against pathogens. Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands .",
"Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets . It has been shown that upon interacting with its receptor, CD40, CD40L induces profound effects on T cells, DCs, B cells, endothelial cells, as well as many cells of the hematopoietic and non-hematopoietic systems. Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin Ig isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy.",
"Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin Ig isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy. It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes . Here we report the successful cloning of the gene encoding cotton rat CD40L crCD40L ; we also expressed and purified the CD40L produced in mammalian cells. Further characterisation of the recombinant cotton rat CD40L revealed its functional activities in promoting DC maturation and cytokine production. weeks old cotton rats were obtained from an inbred colony maintained at Envigo USA .",
"Further characterisation of the recombinant cotton rat CD40L revealed its functional activities in promoting DC maturation and cytokine production. weeks old cotton rats were obtained from an inbred colony maintained at Envigo USA . All animal experiments were conducted in accordance with Institutional Care and Use Committee IACUC of Health Canada Ottawa Animal Care Committee which approved this study. The rats were housed 3 animals per cage in Allentown NexGen individually ventilated cages with free access to food and water. These cages provided a floor space of 142 in 2 / 916 cm 2 . Body weight and any sign of distress were monitored daily. If anything associated the animal health was observed, a full examination would be conducted.",
"Body weight and any sign of distress were monitored daily. If anything associated the animal health was observed, a full examination would be conducted. As In this study spleen cells from normal, healthy animals were isolated, we did not observe any adverse reaction. To isolate splenocytes from the animals, isoflourane was used to put the animals to sleep via inhalation with oxygen for euthanasia. The spleens from three naïve cotton rats were removed aseptically and snap frozen in liquid nitrogen. The spleens were homogenized individually with a TissueLyser II Qiagen and total RNA extracted using the RNeasy Mini kit Qiagen with on-column DNase digestion according to the user's manual.",
"The spleens from three naïve cotton rats were removed aseptically and snap frozen in liquid nitrogen. The spleens were homogenized individually with a TissueLyser II Qiagen and total RNA extracted using the RNeasy Mini kit Qiagen with on-column DNase digestion according to the user's manual. The 3' RACE system Life Technologies was then used with to amplify the 3' portion of the cotton rat CD40L from the total RNA according to the manufacturer's instructions. A schematic of the 3' RACE procedure used is provided in S1 Fig. A gene specific primer 5'-GGACTCTATTATGTCTACACCCAAGTCACCTTCTG -3' was derived from a consensus sequence aligning the rat Rattus norvegicus UniProt: Q9Z2V2 , mouse Mus musculus UniProt: P27548 , and golden hamster Mesocricetus auratus NCBI Reference Sequence: XM_005084522.3 CD40L sequences obtained from the National Center for Biotechnology Information NCBI . Following first strand cDNA synthesis, the 3' portion of the cotton rat CD40L mRNA was PCR amplified using the consensus sequence derived gene specific primer and the abridged universal amplification primer with an annealing temperature at 56˚C.",
"A gene specific primer 5'-GGACTCTATTATGTCTACACCCAAGTCACCTTCTG -3' was derived from a consensus sequence aligning the rat Rattus norvegicus UniProt: Q9Z2V2 , mouse Mus musculus UniProt: P27548 , and golden hamster Mesocricetus auratus NCBI Reference Sequence: XM_005084522.3 CD40L sequences obtained from the National Center for Biotechnology Information NCBI . Following first strand cDNA synthesis, the 3' portion of the cotton rat CD40L mRNA was PCR amplified using the consensus sequence derived gene specific primer and the abridged universal amplification primer with an annealing temperature at 56˚C. The reverse complementary sequence of this primer was then used as a reverse primer with the forward primer 5'-GATAGAAACATACAGCCAACCTTCTCCCAGATC -3' to amplify the 5' portion of the cotton rat CD40L mRNA with an annealing temperature of 57˚C. All amplified fragments were sequenced with BigDye Terminator v.3.1 Cycle Sequencing kit ThermoFisher cat # 4336917 . Briefly, samples were amplified in a PTC-200 thermal cycle MJ Research with the following program: 26 cycles of 1˚C/S to 96˚C, 96˚C for 10 seconds, 1˚C/S to 50˚C, 50˚C for 5 seconds, 1˚C/S to 60˚C, 60˚C for 4 minutes. The samples were cleaned using DyeEx 2.0 Spin kit Qiagen cat # 63204 and loaded onto a 3130xl Genetic Analyzer Applied Biosystems .",
"Briefly, samples were amplified in a PTC-200 thermal cycle MJ Research with the following program: 26 cycles of 1˚C/S to 96˚C, 96˚C for 10 seconds, 1˚C/S to 50˚C, 50˚C for 5 seconds, 1˚C/S to 60˚C, 60˚C for 4 minutes. The samples were cleaned using DyeEx 2.0 Spin kit Qiagen cat # 63204 and loaded onto a 3130xl Genetic Analyzer Applied Biosystems . Raw sequencing data was edited by the instrument's software ThermoFisher 3130xl Genetic Analyzer Data Collection Software v3.0 , and then imported into GeneCodes Sequencher v4.6.1 sequencing analysis software for further editing. The final sequenced contigs are then imported to NCBI BLAST cgi to confirm the identity. Putative conserved domains, trimer interface, and receptor binding sites were determined by performing a standard protein BLAST blastp algorithm; The sequences producing significant alignments were imported into Geneosis software, Auckland, New Zealand . Multiple alignment was conducted as previously described , with phylogenetic analysis using Geneosis Pro 5.6.7.",
"Putative conserved domains, trimer interface, and receptor binding sites were determined by performing a standard protein BLAST blastp algorithm; The sequences producing significant alignments were imported into Geneosis software, Auckland, New Zealand . Multiple alignment was conducted as previously described , with phylogenetic analysis using Geneosis Pro 5.6.7. Once the mRNA sequence was confirmed, a construct was designed beginning with a kozak sequence 5'-CACCGCCGCCACC-3' , followed by a secretion signal consisting of 23 amino acid aa MLLAVLYCLLWSFQTSAGHFPRA from the human tyrosinase signal peptide as previously described . This is followed by six histidine residues to facilitate protein purification. Following this sequence, a 27-aa fragment from the bacteriophage T4 fibritin trimerization motif was added and finally connected to the full length 783bp open reading frame ORF of the cotton rat CD40L sequence at the C terminus. This construct was synthesized and cloned into pUC57 Biobasic, Markham, ON .",
"Following this sequence, a 27-aa fragment from the bacteriophage T4 fibritin trimerization motif was added and finally connected to the full length 783bp open reading frame ORF of the cotton rat CD40L sequence at the C terminus. This construct was synthesized and cloned into pUC57 Biobasic, Markham, ON . Generation of a recombinant vaccinia virus expressing cotton rat CD40L protein construct was achieved using a vaccinia virus E3L and K3L double deletion mutant virus as the parental virus and taterapoxvirus K3L as the positive selection marker Jingxin Cao, unpublished information . Briefly, the recombination plasmid vector for expression of the CD40L construct gene consists of the homologous flanking vaccinia DNA sequences targeting vaccinia A45R gene SOD homolog ; the CD40L construct gene driven by a modified vaccinia H5 promoter Vaccine 1996, 14:1451 , and taterapoxvirus 037 gene driven by vaccinia K3L promoter as the positive selection marker. The recombination vector was transfected into a HeLa PKR knockout cells infected with a vaccinia virus with both E3L and K3L genes deleted. Selection and purification of the recombinant vaccinia virus expressing the CD40L was done in BHK21 cells.",
"The recombination vector was transfected into a HeLa PKR knockout cells infected with a vaccinia virus with both E3L and K3L genes deleted. Selection and purification of the recombinant vaccinia virus expressing the CD40L was done in BHK21 cells. Expression of the CD40L protein was confirmed by Western blotting using His-tag Ab. Cell monolayers were lysed in sample buffer and homogenized using QIAshredder columns Qiagen . Western blotting was performed using 4 to 15% TGX gel and Tris/Glycine/SDS running buffer Bio-Rad Laboratories Inc. , and the protein samples were transferred to Immobilon-FL PVDF membranes Millipore . Protein was detected with Tetra-HIS Ab Qiagen and goat anti-mouse IRDye-800CW LiCor . Membranes were developed using the Odyssey system LiCor .",
"Protein was detected with Tetra-HIS Ab Qiagen and goat anti-mouse IRDye-800CW LiCor . Membranes were developed using the Odyssey system LiCor . The vaccinia virus carrying the crCD40L gene was propagated in BHK21 cells. The cells were collected and washed with PBS once and then lysed with a denaturing buffer 10 mM Tris-HCl, 100 mM sodium phosphate, 6 M guanidine hydrochloride, 10 mM reduced glutathione, pH 8.0 and disrupted by sonication on ice using a Branson sonifier 150 ThermoFisher, Waltham, MA at level 1 for two 10sec bursts with 1min rest on ice between. After separation of cell debris, the supernatant was added to a slurry of Ni-NTA resin Qiagen, Mississauga, ON, Canada 10 mL resin bed and stirred at room temperature for 30 min before loading into a column. The column was purified using an AKTA purifier Amersham Biosciences with Unicorn 5.3 software Amersham Biosciences .",
"After separation of cell debris, the supernatant was added to a slurry of Ni-NTA resin Qiagen, Mississauga, ON, Canada 10 mL resin bed and stirred at room temperature for 30 min before loading into a column. The column was purified using an AKTA purifier Amersham Biosciences with Unicorn 5.3 software Amersham Biosciences . Refolding was accomplished under oxidative conditions with a gradient of denaturing buffer to buffer B buffer B: 10 mM Tris-HCl, 100 mM sodium phosphate, pH 7.8 over 10 column volumes CVs . The column was then washed with three CVs of buffer B + 60 mM imidazole pH 7.8 to remove unspecific binding. The protein was eluted off the column with buffer B + 250 mM imidazole pH 7.8 . The resulting protein was dialysed against PBS pH 7.5 and then confirmed by western blot.",
"The protein was eluted off the column with buffer B + 250 mM imidazole pH 7.8 . The resulting protein was dialysed against PBS pH 7.5 and then confirmed by western blot. 96-well plates were coated with either recombinant mouse CD40L R&D Systems or the recombinant crCD40L protein 2ug/ml in 100μl PBS. Plates were washed with wash buffer PBS-0.1% tween-20 and then blocked with 200μl/well blocking buffer PBS containing 0.1% Tween 20 and 3%IgG Free BSA for 1 hour at 37˚C. Plates were washed with wash buffer and incubated at 37˚C for 1 hour with 100μl/well goat anti-mouseCD40L R&D Systems 2ug/ml in blocking buffer. Plates were subsequently washed and incubated at 37˚C for 1 hour with 100μl/well with rabbit anti-goat IgG HRP conjugate Zymed .",
"Plates were washed with wash buffer and incubated at 37˚C for 1 hour with 100μl/well goat anti-mouseCD40L R&D Systems 2ug/ml in blocking buffer. Plates were subsequently washed and incubated at 37˚C for 1 hour with 100μl/well with rabbit anti-goat IgG HRP conjugate Zymed . Plates were washed again and incubated for 10 min in the dark with 100μl/well 3,3'5,5'-tetramethylbenzidine substrate New England Bio Labs . The reaction was stopped with Stop solution New England Bio Labs and absorbance was read at 450nm on a BioTek Synergy 2 plate reader. Primary bone marrow cells from Balb/c mice Chicago, IL were thawed and cultured in dendritic cell medium from manufacture Cell Biologics M7711 supplemented with GMCSF Cell Biologics without IL-4 at 4x10 5 cells/well in a volume of 200μl. The cells were treated with 0.5μg/ml recombinant mouse CD40L Preprotech, Montreal, QC or the recombinant crCD40L protein at 0.5μg/ml, 5μg/ml, or 50μg/ml.",
"Primary bone marrow cells from Balb/c mice Chicago, IL were thawed and cultured in dendritic cell medium from manufacture Cell Biologics M7711 supplemented with GMCSF Cell Biologics without IL-4 at 4x10 5 cells/well in a volume of 200μl. The cells were treated with 0.5μg/ml recombinant mouse CD40L Preprotech, Montreal, QC or the recombinant crCD40L protein at 0.5μg/ml, 5μg/ml, or 50μg/ml. Forty hours later, flow cytometry was performed on a BD LSRFortessa cell analyser after 2 x 10 5 cells/tube were stained using CD11c-PE-CF594, CD54-FITC, CD40-BV786, CD80-BV421, and CD86-BV711 antibodies. All antibodies were purchased from BD Biosciences. The resulting spectra were analysed using FACS-Diva version 8.0.1 software. To assess IL-6 mRNA production of immature bone marrow murine DCs in response to targeting by recombinant crCD40L, quantitative real-time PCR was conducted on an ABI Prism 7500 Fast Sequence detection system Applied Biosystems .",
"The resulting spectra were analysed using FACS-Diva version 8.0.1 software. To assess IL-6 mRNA production of immature bone marrow murine DCs in response to targeting by recombinant crCD40L, quantitative real-time PCR was conducted on an ABI Prism 7500 Fast Sequence detection system Applied Biosystems . TaqMan assay reagent kits Applied Biosystems were used that contain pre-standardized primers and TaqMan MGB probes for IL-6 and 18S which were used to normalize the data. Total RNA was isolated from 8x10 5 stimulated bone marrow DCs using the RNeasy Mini Kit Qiagen according to manufactures instructions. The isolated RNA was used to make cDNA using the Superscript III First-Strand Synthesis System for RT-PCR Invitrogen according to manufacturer's instructions. The cDNA was then subjected to quantitative PCR using the TaqMan Fast Advanced Master Mix Applied Biosystems according to manufactures instructions.",
"The isolated RNA was used to make cDNA using the Superscript III First-Strand Synthesis System for RT-PCR Invitrogen according to manufacturer's instructions. The cDNA was then subjected to quantitative PCR using the TaqMan Fast Advanced Master Mix Applied Biosystems according to manufactures instructions. Samples were run in duplicate and C t values were obtained. Fold change over unstimulated DCs was calculated using the 2 -ΔΔCT method of relative quantification , using 18S as the housekeeping reference gene. To investigate IL-6 secretion by murine bone marrow DCs, supernatant from forty hour stimulated cultures were collected and assayed using the Mouse IL-6 DuoSet ELISA Kit R & D Systems following the manufacturer's protocol. The complete mRNA sequence of CD40L was obtained in two steps Fig 1 .",
"To investigate IL-6 secretion by murine bone marrow DCs, supernatant from forty hour stimulated cultures were collected and assayed using the Mouse IL-6 DuoSet ELISA Kit R & D Systems following the manufacturer's protocol. The complete mRNA sequence of CD40L was obtained in two steps Fig 1 . A sequence corresponding to nucleotides 535 through to the poly-A tail was obtained using the 3' RACE kit and mRNA as starting material, which was isolated from cotton rat splenocytes and a rodent consensus sequence as a primer. This portion of the sequence has the 3' un-translated region of the mRNA as well as the stop codon. The 5' end of the protein was obtained in the next step by PCR amplification of the cDNA obtained in the first step with the 3' RACE kit and the reverse complement of the consensus sequence primer and a second consensus sequence primer designed to bind to the beginning of the CD40L mRNA. The 783bp ORF encodes 260aa followed by a stop codon.",
"The 5' end of the protein was obtained in the next step by PCR amplification of the cDNA obtained in the first step with the 3' RACE kit and the reverse complement of the consensus sequence primer and a second consensus sequence primer designed to bind to the beginning of the CD40L mRNA. The 783bp ORF encodes 260aa followed by a stop codon. Comparison of the sequenced CD40L gene revealed that the crCD40L coding sequence shares 93%, 89%, and 83%, identity with golden hamster, rat, and mouse, respectively. At the amino acid aa level, the corresponding identities are 91%, 82%, and 82%, Fig 2a. At both the mRNA and aa levels, the crCD40L shared the closest similarity with Peromyscus maniculatus bairdii or deer mouse at 93% and 92% respectively. When sequence homology analysis is performed, crCD40L clusters with other members of the Cricetidae family Fig 2b.",
"At both the mRNA and aa levels, the crCD40L shared the closest similarity with Peromyscus maniculatus bairdii or deer mouse at 93% and 92% respectively. When sequence homology analysis is performed, crCD40L clusters with other members of the Cricetidae family Fig 2b. We next examined the functional domains in crCD40L in comparison with other known CD40L. As shown in Fig 3a, crCD40L has a putative tumor necrosis factor TNF superfamily Using EZmol software , we predicted folding of the protein as shown in Fig 3b. The cotton rat CD40L cDNA that we have isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783bp which coded for a 260 aa protein. The homology of cotton rat CD40L, at both the amino acid and nucleic acid level, is closer to members of the Cricetidae family hamster and deer mouse than to those of the Muridae family rat and mouse as shown in Fig 2b.",
"The cotton rat CD40L cDNA that we have isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783bp which coded for a 260 aa protein. The homology of cotton rat CD40L, at both the amino acid and nucleic acid level, is closer to members of the Cricetidae family hamster and deer mouse than to those of the Muridae family rat and mouse as shown in Fig 2b. As with other known CD40L proteins, there is a putative TNF superfamily domain, a transmembrane domain, trimerization sites, and receptor binding sites . TNF superfamily members include TNF TNF-alpha , LT lymphotoxin-alpha, TNF-beta , CD40 ligand, Apo2L TRAIL , Fas ligand, and osteoprotegerin OPG ligand, among others . The TNF superfamily is composed of 19 ligands and 29 receptors, in which each has vastly diversified roles in the body and exhibit pro-inflammatory activity, partly via activation of NF-kB . Members of this family generally have an intracellular N-terminal domain, a short transmembrane segment, an extracellular stalk, and a globular TNF-like extracellular domain of about 150 residues .",
"The TNF superfamily is composed of 19 ligands and 29 receptors, in which each has vastly diversified roles in the body and exhibit pro-inflammatory activity, partly via activation of NF-kB . Members of this family generally have an intracellular N-terminal domain, a short transmembrane segment, an extracellular stalk, and a globular TNF-like extracellular domain of about 150 residues . They initiate apoptosis by binding to related receptors, some of which have intracellular death domains . These proteins typically form homo-or hetero-trimeric complexes and bind one elongated receptor molecule along each of three clefts formed by neighboring monomers of the trimer and ligand trimerization is for receptor binding . All seven known conserved residues that constitute the trimer interface on the conserved TNF domain , were mapped to the putative crCD40L protein sequence. Additionally, all six known conserved receptor binding sites on the conserved TNF domain , were mapped to the crCD40L protein sequence.",
"All seven known conserved residues that constitute the trimer interface on the conserved TNF domain , were mapped to the putative crCD40L protein sequence. Additionally, all six known conserved receptor binding sites on the conserved TNF domain , were mapped to the crCD40L protein sequence. In order to further evaluate the crCD40L deduced sequence, the full 783bp ORF of the crCD40L was cloned into a vaccinia virus vector. The crCD40L construct was designed to carry a secretion signal, histidine tag, and a trimerization motif Fig 4a . Selection and purification of the recombinant vaccinia virus expressing the CD40L construct was conducted in BHK21 cells. Western blot with anti-histidine antibody Ab was used to confirm expression of the CD40L protein construct Fig 4b and S2 Fig.",
"Selection and purification of the recombinant vaccinia virus expressing the CD40L construct was conducted in BHK21 cells. Western blot with anti-histidine antibody Ab was used to confirm expression of the CD40L protein construct Fig 4b and S2 Fig. The resulting 36 kDa protein product was found in both the cell lysate and supernatant faint band-48 hours only . Since the highest expression was found in the cell lysate, it was used for further purification of the protein. It should be noted that the protein was only able to be detected under reducing conditions. Under non-reducing conditions, the protein was unable to be detected by the anti-histidine Ab, even in the cell lysate data not shown .",
"It should be noted that the protein was only able to be detected under reducing conditions. Under non-reducing conditions, the protein was unable to be detected by the anti-histidine Ab, even in the cell lysate data not shown . This indicates that the histidine tag is folded within the trimer and is unavailable in the native form for purification. This is an additional reason for the need to purify the protein from the cell lysate under harsh denaturing conditions followed by protein refolding. The reason we utilized a mammalian expression system to produce the protein rather than a bacterial system is to facilitate its proper folding into its native structure, trimerization, and glycosylation. The aa backbone predicts a protein of 29 kDa, yet initial studies of the CD40L protein suggested a molecular mass of 39 kDa, and on most cell types the molecular mass of CD40L is 32-33kDa, consistent with extensive post-translation modification .",
"The reason we utilized a mammalian expression system to produce the protein rather than a bacterial system is to facilitate its proper folding into its native structure, trimerization, and glycosylation. The aa backbone predicts a protein of 29 kDa, yet initial studies of the CD40L protein suggested a molecular mass of 39 kDa, and on most cell types the molecular mass of CD40L is 32-33kDa, consistent with extensive post-translation modification . The BHK21 cells expressing the crCD40L construct were collected and lysed with 6 M guanidine hydrochloride with reduced glutathione and sonication. The lysate was loaded on the nickel column and the washed with denaturing buffer as described in materials and methods. The bound proteins were refolded on the column with gradient buffer exchange, to allow slow refold the protein, given that CD40L biological activity is dependent on a homo-trimer configuration . The resulting bound protein was subsequently eluted with imidazole.",
"The bound proteins were refolded on the column with gradient buffer exchange, to allow slow refold the protein, given that CD40L biological activity is dependent on a homo-trimer configuration . The resulting bound protein was subsequently eluted with imidazole. The resulting fractions that showed a peak were pooled and dialysed against PBS. The purified protein was confirmed in ELISA. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence, an Ab known to detect mouse CD40L was used to identify the purified crCD40L protein. The purified recombinant crCD40L was used as a coating antigen in a concentration gradient manner, and was detected with an Ab generated against the mouse CD40L at all concentrations Fig 5 .",
"Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence, an Ab known to detect mouse CD40L was used to identify the purified crCD40L protein. The purified recombinant crCD40L was used as a coating antigen in a concentration gradient manner, and was detected with an Ab generated against the mouse CD40L at all concentrations Fig 5 . Uncoated controls were performed in parallel and were negative for CD40L in ELISA. We measured the overall strength of the antigen-antibody complex in the presence of 6M urea . The avidity of the cotton rat CD40L for the anti-mouse CD40L antibody was decreased in the presence of 6M urea at all concentrations. Clearly, as the antibody used was raised against mouse CD40L, the crCD40L is detected by mouse CD40L.",
"The avidity of the cotton rat CD40L for the anti-mouse CD40L antibody was decreased in the presence of 6M urea at all concentrations. Clearly, as the antibody used was raised against mouse CD40L, the crCD40L is detected by mouse CD40L. crCD40L was expressed in vaccinia virus and purified from infected BHK21 cell lysate on a nickel column. The purified protein was detected by ELISA using a mouse antibody against CD40L in a concentration gradient dependent manner. The avidity of the mouse CD40L antibody to the cotton rat CD40L protein was evaluated in the presence of 6M urea. The difference between the untreated and 6M urea treated for each group was calculated using students t-test ÃÃÃ p<0.001, ÃÃÃÃ p<0.0001 n = 2 .",
"The avidity of the mouse CD40L antibody to the cotton rat CD40L protein was evaluated in the presence of 6M urea. The difference between the untreated and 6M urea treated for each group was calculated using students t-test ÃÃÃ p<0.001, ÃÃÃÃ p<0.0001 n = 2 . Data shown is a representative experiment of three separate experiments where two n = 2 technical replicates are conducted in each experiment. The no-coating and noprimary antibody negative controls gave average OD values of 0.56 and 0.107 respectively. addition of urea treatment would substantially weaken the interaction between the antibody and crCD40L. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence with similar functional domains, we evaluated the biological activity of the recombinant crCD40L on immature murine bone marrow DCs.",
"addition of urea treatment would substantially weaken the interaction between the antibody and crCD40L. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence with similar functional domains, we evaluated the biological activity of the recombinant crCD40L on immature murine bone marrow DCs. We conducted experiments based on known functional activities of CD40L in other animal species. Specifically, maturation of immature DCs after exposure to antigen is known to play a crucial role in their immunity-stimulating function , while trimeric recombinant CD40L has been shown to stimulate DC immunomodulating functions . When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated .",
"When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L . Moreover, mouse I-A d major histocompatibility complex is also up-regulated upon stimulation with CD40L . When our recombinant crCD40L was used to stimulate immature murine bone marrow DCs, we observed similar results to that when murine CD40L is used Tables 1 and 2 . CD11c was down regulated in both median flouresence intensity Table 1 and the percentage of positive cells Table 2 .",
"When our recombinant crCD40L was used to stimulate immature murine bone marrow DCs, we observed similar results to that when murine CD40L is used Tables 1 and 2 . CD11c was down regulated in both median flouresence intensity Table 1 and the percentage of positive cells Table 2 . The co-stimulatory molecules CD54, CD40, CD80, and CD86 were all up-regulated in both median fluorescence intensity Table 1 and the percentage of positive cells Table 2 . The Mouse I-A d major histocompatibility complex was upregulated in median fluorescence intensity Table 1 but not up-regulated in terms of the overall percentage of positive cells Table 2 . We speculate this to be due to the species incompatibility since we are stimulating mouse bone marrow cells with cotton rat CD40L. Nevertheless, the crCD40L was able to promote up-regulation of key co-stimulatory markers on immature DCs promoting DC maturation.",
"We speculate this to be due to the species incompatibility since we are stimulating mouse bone marrow cells with cotton rat CD40L. Nevertheless, the crCD40L was able to promote up-regulation of key co-stimulatory markers on immature DCs promoting DC maturation. The gating strategy used for the flow cytometry analysis is provided in S3 Fig along with overlapping histograms of the intracellular adhesion marker and co-stimulatory markers. CD40-induced activation of cytokine gene expression in DCs by CD40L is an important process in the initiation of primary immune responses and is critical for DC maturation and the generation of antigen-specific T cell responses . IL-6 is a highly pleiotropic cytokine in that it stimulates the activation, proliferation, and survival of T cells, and furthermore, modifies DC function and survival . We tested if the recombinant crCD40L could induce IL-6 gene expression Fig 6a and production of the cytokine Fig 6b by immature murine bone marrow DCs.",
"IL-6 is a highly pleiotropic cytokine in that it stimulates the activation, proliferation, and survival of T cells, and furthermore, modifies DC function and survival . We tested if the recombinant crCD40L could induce IL-6 gene expression Fig 6a and production of the cytokine Fig 6b by immature murine bone marrow DCs. The results indicate that a significant increase in both IL-6 gene expression and cytokine production in immature murine bone marrow DCs was observed forty hours after stimulation with the crCD40L. Collectively, the observation that both the upregulation of immature DC cell surface maturation markers and increased IL-6 gene expression and cytokine production provide strong evidence of the biological activity of crCD40L. In summary, the cotton rat CD40L cDNA that we isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783 bp which coded for a 260 aa protein. The recombinant cotton rat CD40L was recognized by an Ab against mouse CD40L in direct ELISA, and showed biological activity by upregulating maturation markers CD40, CD54, CD80, and CD86 as well as I-A d on immature bone marrow murine DCs and moreover, inducing upregulation of IL-6 gene and cytokine expression in these cells.",
"In summary, the cotton rat CD40L cDNA that we isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783 bp which coded for a 260 aa protein. The recombinant cotton rat CD40L was recognized by an Ab against mouse CD40L in direct ELISA, and showed biological activity by upregulating maturation markers CD40, CD54, CD80, and CD86 as well as I-A d on immature bone marrow murine DCs and moreover, inducing upregulation of IL-6 gene and cytokine expression in these cells. The isolation of the cotton rat CD40L sequence and availability of CD40L has the potential to positively impact basic immunological research and vaccine development, given the critical importance of this protein in orchestrating immune responses ."
] | 1,570 | 1,634 |
What factor may influence viral replication and gene expression?
|
the average codon usage frequencies in the host genome
|
[
"BACKGROUND: Rabbit haemorrhagic disease virus RHDV , as the pathogeny of Rabbit haemorrhagic disease, can cause a highly infectious and often fatal disease only affecting wild and domestic rabbits. Recent researches revealed that it, as one number of the Caliciviridae, has some specialties in its genome, its reproduction and so on. RESULTS: In this report, we firstly analyzed its genome and two open reading frameworks ORFs from this aspect of codon usage bias. Our researches indicated that mutation pressure rather than natural is the most important determinant in RHDV with high codon bias, and the codon usage bias is nearly contrary between ORF1 and ORF2, which is maybe one of factors regulating the expression of VP60 encoding by ORF1 and VP10 encoding by ORF2 . Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. CONCLUSIONS: We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV.",
"Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. CONCLUSIONS: We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV. Text: Synonymous codons are not used randomly . The variation of codon usage among ORFs in different organisms is accounted by mutational pressure and translational selection as two main factors . Levels and causes of codon usage bias are available to understand viral evolution and the interplay between viruses and the immune response .",
"The variation of codon usage among ORFs in different organisms is accounted by mutational pressure and translational selection as two main factors . Levels and causes of codon usage bias are available to understand viral evolution and the interplay between viruses and the immune response . Thus, many organisms such as bacteria, yeast, Drosophila, and mammals, have been studied in great detail up on codon usage bias and nucleotide composition . However, same researches in viruses, especially in animal viruses, have been less studied. It has been observed that codon usage bias in human RNA viruses is related to mutational pressure, G +C content, the segmented nature of the genome and the route of transmission of the virus . For some vertebrate DNA viruses, genome-wide mutational pressure is regarded as the main determinant of codon usage rather than natural selection for specific coding triplets .",
"It has been observed that codon usage bias in human RNA viruses is related to mutational pressure, G +C content, the segmented nature of the genome and the route of transmission of the virus . For some vertebrate DNA viruses, genome-wide mutational pressure is regarded as the main determinant of codon usage rather than natural selection for specific coding triplets . Analysis of the bovine papillomavirus type 1 BPV1 late genes has revealed a relationship between codon usage and tRNA availability . In the mammalian papillomaviruses, it has been proposed that differences from the average codon usage frequencies in the host genome strongly influence both viral replication and gene expression . Codon usage may play a key role in regulating latent versus productive infection in Epstein-Barr virus . Recently, it was reported that codon usage is an important driving force in the evolution of astroviruses and small DNA viruses .",
"Codon usage may play a key role in regulating latent versus productive infection in Epstein-Barr virus . Recently, it was reported that codon usage is an important driving force in the evolution of astroviruses and small DNA viruses . Clearly, studies of synonymous codon usage in viruses can reveal much about the molecular evolution of viruses or individual genes. Such information would be relevant in understanding the regulation of viral gene expression. Up to now, little codon usage analysis has been performed on Rabbit haemorrhagic disease virus RHDV , which is the pathogen causing Rabbit haemorrhagic disease RHD , also known as rabbit calicivirus disease RCD or viral haemorrhagic disease VHD , a highly infectious and often fatal disease that affects wild and domestic rabbits. Although the virus infects only rabbits, RHD continues to cause serious problems in different parts of the world.",
"Up to now, little codon usage analysis has been performed on Rabbit haemorrhagic disease virus RHDV , which is the pathogen causing Rabbit haemorrhagic disease RHD , also known as rabbit calicivirus disease RCD or viral haemorrhagic disease VHD , a highly infectious and often fatal disease that affects wild and domestic rabbits. Although the virus infects only rabbits, RHD continues to cause serious problems in different parts of the world. RHDV is a single positive stranded RNA virus without envelope, which contains two open reading frames ORFs separately encoding a predicted polyprotein and a minor structural protein named VP10 . After the hydrolysis of self-coding 3C-like cysteinase, the polyprotein was finally hydrolyzed into 8 cleavage products including 7 nonstructural proteins and 1 structural protein named as VP60 . Studies on the phylogenetic relationship of RHDVs showed only one serotype had been isolated, and no genotyping for RHDV was reported. It reported that the VP10 was translated with an efficiency of 20% of the preceding ORF1 .",
"Studies on the phylogenetic relationship of RHDVs showed only one serotype had been isolated, and no genotyping for RHDV was reported. It reported that the VP10 was translated with an efficiency of 20% of the preceding ORF1 . In order to better understand the characteristics of the RHDV genome and to reveal more information about the viral genome, we have analyzed the codon usage and dinucleotide composition. In this report, we sought to address the following issues concerning codon usage in RHDV: i the extent and causes of codon bias in RHDV; ii A possible genotyping of RHDV; iii Codon usage bias as a factor reducing the expression of VP10 and iiii the evolution of the ORFs. The 30 available complete RNA sequences of RHDV were obtained from GenBank randomly in January 2011. The serial number SN , collection dates, isolated areas and GenBank accession numbers are listed in Table 1 .",
"The 30 available complete RNA sequences of RHDV were obtained from GenBank randomly in January 2011. The serial number SN , collection dates, isolated areas and GenBank accession numbers are listed in Table 1 . To investigate the characteristics of synonymous codon usage without the influence of amino acid composition, RSCU values of each codon in a ORF of RHDV were calculated according to previous reports 2 Sharp, Tuohy et al. 1986 as the followed formula: Where g ij is the observed number of the ith codon for jth amino acid which has n i type of synonymous codons. The codons with RSCU value higher than 1.0 have positive codon usage bias, while codons with value lower than 1.0 has relative negative codon usage bias. As RSCU values of some codons are nearly equal to 1.0, it means that these codons are chosen equally and randomly.",
"The codons with RSCU value higher than 1.0 have positive codon usage bias, while codons with value lower than 1.0 has relative negative codon usage bias. As RSCU values of some codons are nearly equal to 1.0, it means that these codons are chosen equally and randomly. The index GC3s means the fraction of the nucleotides G+C at the synonymous third codon position, excluding Met, Trp, and the termination codons. The ENC, as the best estimator of absolute synonymous codon usage bias , was calculated for the quantification of the codon usage bias of each ORF . The predicted values of ENC were calculated as ENC = 2 + s + 29 where s represents the given G+C 3 % value. The values of ENC can also be obtained by EMBOSS CHIPS program .",
"The predicted values of ENC were calculated as ENC = 2 + s + 29 where s represents the given G+C 3 % value. The values of ENC can also be obtained by EMBOSS CHIPS program . Analyses were conducted with the Nei-Gojobori model , involving 30 nucleotide sequences. All positions containing gaps and missing data were eliminated. The values of dn, ds and ω dn/ds were calculated in MEGA4.0 . Multivariate statistical analysis can be used to explore the relationships between variables and samples. In this study, correspondence analysis was used to investigate the major trend in codon usage variation among ORFs.",
"Multivariate statistical analysis can be used to explore the relationships between variables and samples. In this study, correspondence analysis was used to investigate the major trend in codon usage variation among ORFs. In this study, the complete coding region of each ORF was represented as a 59 dimensional vector, and each dimension corresponds to the RSCU value of one sense codon excluding Met, Trp, and the termination codons . Correlation analysis was used to identify the relationship between nucleotide composition and synonymous codon usage pattern . This analysis was implemented based on the Spearman's rank correlation analysis way. All statistical processes were carried out by with statistical software SPSS 17.0 for windows.",
"This analysis was implemented based on the Spearman's rank correlation analysis way. All statistical processes were carried out by with statistical software SPSS 17.0 for windows. The values of nucleotide contents in complete coding region of all 30 RHDV genomes were analyzed and listed in Table 2 and Table 3 . Evidently, C+G % content of the ORF1 fluctuated from 50.889 to 51.557 with a mean value of 51.14557, and C+G % content of the ORF2 were ranged from 35.593 to 40.113 with a mean value of 37.6624, which were indicating that nucleotides A and U were the major elements of ORF2 against ORF1. Comparing the values of A 3 %, U 3 %, C 3 % and G 3 %, it is clear that C 3 % was distinctly high and A 3 % was the lowest of all in ORF1 of RHDV, while U 3 % was distinctly high and C 3 % was the lowest of all in ORF2 of Table 2 Identified nucleotide contents in complete coding region length > 250 bps in the ORF1 of RHDV 30 isolates genome Table 4 . Most preferentially used codons in ORF1 were C-ended or G-ended codons except Ala, Pro and Ser, however, A-ended or G-ended codons were preferred as the content of ORF2.",
"Comparing the values of A 3 %, U 3 %, C 3 % and G 3 %, it is clear that C 3 % was distinctly high and A 3 % was the lowest of all in ORF1 of RHDV, while U 3 % was distinctly high and C 3 % was the lowest of all in ORF2 of Table 2 Identified nucleotide contents in complete coding region length > 250 bps in the ORF1 of RHDV 30 isolates genome Table 4 . Most preferentially used codons in ORF1 were C-ended or G-ended codons except Ala, Pro and Ser, however, A-ended or G-ended codons were preferred as the content of ORF2. In addition, the dn, ds and ω dN/dS values of ORF1 were separately 0.014, 0.338 and 0.041, and the values of ORF2 were 0.034, 0.103 and 0.034, respectively. The ω values of two ORFs in RHDV genome are generally low, indicating that the RHDV whole genome is subject to relatively strong selective constraints. COA was used to investigate the major trend in codon usage variation between two ORFs of all 30 RHDV selected for this study. After COA for RHDV Genome, one major trend in the first axis f' 1 which accounted for 42.967% of the total variation, and another major trend in the second axis f' 2 which accounted for 3.632% of the total variation.",
"COA was used to investigate the major trend in codon usage variation between two ORFs of all 30 RHDV selected for this study. After COA for RHDV Genome, one major trend in the first axis f' 1 which accounted for 42.967% of the total variation, and another major trend in the second axis f' 2 which accounted for 3.632% of the total variation. The coordinate of the complete coding region of each ORF was plotted in Figure 1 defining by the first and second principal axes. It is clear that coordinate of each ORF is relatively isolated. Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group marked as Group 1 is To estimate whether the evolution of RHDV genome on codon usage was regulated by mutation pressure or natural selection, the A%, U%, C%, G% and C+G % were compared with A 3 %, U 3 %, C 3 %, G 3 % and C 3 +G 3 %, respectively Table 5 . There is a complex correlation among nucleotide compositions.",
"Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group marked as Group 1 is To estimate whether the evolution of RHDV genome on codon usage was regulated by mutation pressure or natural selection, the A%, U%, C%, G% and C+G % were compared with A 3 %, U 3 %, C 3 %, G 3 % and C 3 +G 3 %, respectively Table 5 . There is a complex correlation among nucleotide compositions. In detail, A 3 %, U 3 %, C 3 % and G 3 % have a significant negative correlation with G%, C%, U% and A% and positive correlation with A%, U%, C% and G%, respectively. It suggests that nucleotide constraint may influence synonymous codon usage patterns. However, A 3 % has non-correlation with U% and C%, and U 3 % has noncorrelation with A% and G%, respectively, which haven't indicated any peculiarity about synonymous codon usage. Furthermore, C 3 % and G 3 % have non-correlation with A%, G% and U%, C%, respectively, indicating these data don't reflect the true feature of synonymous codon usage as well.",
"However, A 3 % has non-correlation with U% and C%, and U 3 % has noncorrelation with A% and G%, respectively, which haven't indicated any peculiarity about synonymous codon usage. Furthermore, C 3 % and G 3 % have non-correlation with A%, G% and U%, C%, respectively, indicating these data don't reflect the true feature of synonymous codon usage as well. Therefore, linear regression analysis was implemented to analyze the correlation between synonymous codon usage bias and nucleotide compositions. Details of correlation analysis between the first two principle axes f' 1 and f' 2 of each RHDV genome in COA and nucleotide contents were listed in Table 6 . In surprise, only f2 values are closely related to base nucleotide A and G content on the third codon position only, suggesting that nucleotide A and G is a factor influencing the synonymous codon usage pattern of RHDV genome. However, f' 1 value has non-correlation with base nucleotide contents on the third codon position; it is observably suggest that codon usage patterns in RHDV were probably influenced by other factors, such as the second structure of viral genome and limits of host.",
"In surprise, only f2 values are closely related to base nucleotide A and G content on the third codon position only, suggesting that nucleotide A and G is a factor influencing the synonymous codon usage pattern of RHDV genome. However, f' 1 value has non-correlation with base nucleotide contents on the third codon position; it is observably suggest that codon usage patterns in RHDV were probably influenced by other factors, such as the second structure of viral genome and limits of host. In spite of that, compositional constraint is a factor shaping the pattern of synonymous codon usage in RHDV genome. Figure 1 A plot of value of the first and second axis of RHDV genome in COA. The first axis f' 1 accounts for 42.967% of the total variation, and the second axis f' 2 accounts for 3.632% of the total variation. Table 5 Summary of correlation analysis between the A, U, C, G contents and A 3 , U 3 , C 3 , G 3 contents in all selected samples There have been more and more features that are unique to RHDV within the family Caliciviridae, including its single host tropism, its genome and its VP10 as a structural protein with unknown function.",
"The first axis f' 1 accounts for 42.967% of the total variation, and the second axis f' 2 accounts for 3.632% of the total variation. Table 5 Summary of correlation analysis between the A, U, C, G contents and A 3 , U 3 , C 3 , G 3 contents in all selected samples There have been more and more features that are unique to RHDV within the family Caliciviridae, including its single host tropism, its genome and its VP10 as a structural protein with unknown function. After we analyzed synonymous codon usage in RHDV Table 2 , we obtained several conclusions and conjectures as followed. 4.1 Mutational bias as a main factor leading to synonymous codon usage variation ENC-plot, as a general strategy, was utilized to investigate patterns of synonymous codon usage. The ENC-plots of ORFs constrained only by a C 3 +G 3 composition will lie on or just below the curve of the predicted values . ENC values of RHDV genomes were plotted against its corresponding C 3 +G 3 %.",
"The ENC-plots of ORFs constrained only by a C 3 +G 3 composition will lie on or just below the curve of the predicted values . ENC values of RHDV genomes were plotted against its corresponding C 3 +G 3 %. All of the spots lie below the curve of the predicted values, as shown in Figure 2 , suggesting that the codon usage bias in all these 30 RHDV genomes is principally influenced by the mutational bias. As we know, the efficiency of gene expression is influenced by regulator sequences or elements and codon usage bias. It reported that the RNA sequence of the 3terminal 84 nucleotides of ORF1were found to be crucial for VP10 expression instead of the encoded peptide. VP10 coding by ORF2 has been reported as a low expressive structural protein against VP60 coding by ORF1 .",
"It reported that the RNA sequence of the 3terminal 84 nucleotides of ORF1were found to be crucial for VP10 expression instead of the encoded peptide. VP10 coding by ORF2 has been reported as a low expressive structural protein against VP60 coding by ORF1 . And its efficiency of translation is only 20% of VP60. According to results showed by Table 4 , it revealed the differences in codon usage patterns of two ORFs, which is a possible factor reducing the expression of VP10. Although VP10 encoded by ORF2, as a minor structural protein with unknown functions, has been described by LIU as a nonessential protein for virus infectivity, the ω Figure 2 Effective number of codons used in each ORF plotted against the GC3s. The continuous curve plots the relationship between GC3s and ENC in the absence of selection.",
"Although VP10 encoded by ORF2, as a minor structural protein with unknown functions, has been described by LIU as a nonessential protein for virus infectivity, the ω Figure 2 Effective number of codons used in each ORF plotted against the GC3s. The continuous curve plots the relationship between GC3s and ENC in the absence of selection. All of spots lie below the expected curve. value of ORF2 suggests VP10 plays an important role in the certain stage of whole RHDV lifecycle. After combining with low expression and ω value of VP10, we conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. This mechanism has been confirmed in various positive-chain RNA viruses, including coxsackievirus, dengue virus, equine arterivirus, footand-mouth disease virus, hepatitis C virus, poliovirus, rhinovirus, and severe acute respiratory syndrome , although the details remain elusive.",
"After combining with low expression and ω value of VP10, we conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. This mechanism has been confirmed in various positive-chain RNA viruses, including coxsackievirus, dengue virus, equine arterivirus, footand-mouth disease virus, hepatitis C virus, poliovirus, rhinovirus, and severe acute respiratory syndrome , although the details remain elusive. As preceding description, ENC reflects the evolution of codon usage variation and nucleotide composition to some degree. After the correlation analysis of ENC values between ORF1 and ORF2 Table 7 , the related coefficient of ENC values of two ORFs is 0.230, and p value is 0.222 more than 0.05. These data revealed that no correlation existed in ENC values of two ORFs, indicating that codon usage patterns and evolution of two ORFs are separated each other. Further, this information maybe helps us well understand why RSCU and ENC between two ORFs are quite different.",
"These data revealed that no correlation existed in ENC values of two ORFs, indicating that codon usage patterns and evolution of two ORFs are separated each other. Further, this information maybe helps us well understand why RSCU and ENC between two ORFs are quite different. Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group marked as Group 1 is positive value and the other one marked as Group 2 is negative value. And all of those strains isolated before 2000 belonged to Group 2, including Italy-90, RHDV-V351, RHDV-FRG, BS89, RHDV-SD and M67473.1. Although RHDV has been reported as only one type, this may be a reference on dividing into two genotypes. In this report, we firstly analyzed its genome and two open reading frameworks ORFs from this aspect of codon usage bias.",
"Although RHDV has been reported as only one type, this may be a reference on dividing into two genotypes. In this report, we firstly analyzed its genome and two open reading frameworks ORFs from this aspect of codon usage bias. Our researches indicated that mutation pressure rather than natural is the most important determinant in RHDV with high codon bias, and the codon usage bias is nearly contrary between ORF1 and ORF2, which is maybe one of factors regulating the expression of VP60 encoding by ORF1 and VP10 encoding by ORF2 . Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV.",
"We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV. All the results will guide the next researches on the RHDV as a reference."
] | 1,567 | 567 |
What accounts for the variation of codon usage among open reading frameworks?
|
mutational pressure and translational selection
|
[
"BACKGROUND: Rabbit haemorrhagic disease virus RHDV , as the pathogeny of Rabbit haemorrhagic disease, can cause a highly infectious and often fatal disease only affecting wild and domestic rabbits. Recent researches revealed that it, as one number of the Caliciviridae, has some specialties in its genome, its reproduction and so on. RESULTS: In this report, we firstly analyzed its genome and two open reading frameworks ORFs from this aspect of codon usage bias. Our researches indicated that mutation pressure rather than natural is the most important determinant in RHDV with high codon bias, and the codon usage bias is nearly contrary between ORF1 and ORF2, which is maybe one of factors regulating the expression of VP60 encoding by ORF1 and VP10 encoding by ORF2 . Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. CONCLUSIONS: We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV.",
"Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. CONCLUSIONS: We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV. Text: Synonymous codons are not used randomly . The variation of codon usage among ORFs in different organisms is accounted by mutational pressure and translational selection as two main factors . Levels and causes of codon usage bias are available to understand viral evolution and the interplay between viruses and the immune response .",
"The variation of codon usage among ORFs in different organisms is accounted by mutational pressure and translational selection as two main factors . Levels and causes of codon usage bias are available to understand viral evolution and the interplay between viruses and the immune response . Thus, many organisms such as bacteria, yeast, Drosophila, and mammals, have been studied in great detail up on codon usage bias and nucleotide composition . However, same researches in viruses, especially in animal viruses, have been less studied. It has been observed that codon usage bias in human RNA viruses is related to mutational pressure, G +C content, the segmented nature of the genome and the route of transmission of the virus . For some vertebrate DNA viruses, genome-wide mutational pressure is regarded as the main determinant of codon usage rather than natural selection for specific coding triplets .",
"It has been observed that codon usage bias in human RNA viruses is related to mutational pressure, G +C content, the segmented nature of the genome and the route of transmission of the virus . For some vertebrate DNA viruses, genome-wide mutational pressure is regarded as the main determinant of codon usage rather than natural selection for specific coding triplets . Analysis of the bovine papillomavirus type 1 BPV1 late genes has revealed a relationship between codon usage and tRNA availability . In the mammalian papillomaviruses, it has been proposed that differences from the average codon usage frequencies in the host genome strongly influence both viral replication and gene expression . Codon usage may play a key role in regulating latent versus productive infection in Epstein-Barr virus . Recently, it was reported that codon usage is an important driving force in the evolution of astroviruses and small DNA viruses .",
"Codon usage may play a key role in regulating latent versus productive infection in Epstein-Barr virus . Recently, it was reported that codon usage is an important driving force in the evolution of astroviruses and small DNA viruses . Clearly, studies of synonymous codon usage in viruses can reveal much about the molecular evolution of viruses or individual genes. Such information would be relevant in understanding the regulation of viral gene expression. Up to now, little codon usage analysis has been performed on Rabbit haemorrhagic disease virus RHDV , which is the pathogen causing Rabbit haemorrhagic disease RHD , also known as rabbit calicivirus disease RCD or viral haemorrhagic disease VHD , a highly infectious and often fatal disease that affects wild and domestic rabbits. Although the virus infects only rabbits, RHD continues to cause serious problems in different parts of the world.",
"Up to now, little codon usage analysis has been performed on Rabbit haemorrhagic disease virus RHDV , which is the pathogen causing Rabbit haemorrhagic disease RHD , also known as rabbit calicivirus disease RCD or viral haemorrhagic disease VHD , a highly infectious and often fatal disease that affects wild and domestic rabbits. Although the virus infects only rabbits, RHD continues to cause serious problems in different parts of the world. RHDV is a single positive stranded RNA virus without envelope, which contains two open reading frames ORFs separately encoding a predicted polyprotein and a minor structural protein named VP10 . After the hydrolysis of self-coding 3C-like cysteinase, the polyprotein was finally hydrolyzed into 8 cleavage products including 7 nonstructural proteins and 1 structural protein named as VP60 . Studies on the phylogenetic relationship of RHDVs showed only one serotype had been isolated, and no genotyping for RHDV was reported. It reported that the VP10 was translated with an efficiency of 20% of the preceding ORF1 .",
"Studies on the phylogenetic relationship of RHDVs showed only one serotype had been isolated, and no genotyping for RHDV was reported. It reported that the VP10 was translated with an efficiency of 20% of the preceding ORF1 . In order to better understand the characteristics of the RHDV genome and to reveal more information about the viral genome, we have analyzed the codon usage and dinucleotide composition. In this report, we sought to address the following issues concerning codon usage in RHDV: i the extent and causes of codon bias in RHDV; ii A possible genotyping of RHDV; iii Codon usage bias as a factor reducing the expression of VP10 and iiii the evolution of the ORFs. The 30 available complete RNA sequences of RHDV were obtained from GenBank randomly in January 2011. The serial number SN , collection dates, isolated areas and GenBank accession numbers are listed in Table 1 .",
"The 30 available complete RNA sequences of RHDV were obtained from GenBank randomly in January 2011. The serial number SN , collection dates, isolated areas and GenBank accession numbers are listed in Table 1 . To investigate the characteristics of synonymous codon usage without the influence of amino acid composition, RSCU values of each codon in a ORF of RHDV were calculated according to previous reports 2 Sharp, Tuohy et al. 1986 as the followed formula: Where g ij is the observed number of the ith codon for jth amino acid which has n i type of synonymous codons. The codons with RSCU value higher than 1.0 have positive codon usage bias, while codons with value lower than 1.0 has relative negative codon usage bias. As RSCU values of some codons are nearly equal to 1.0, it means that these codons are chosen equally and randomly.",
"The codons with RSCU value higher than 1.0 have positive codon usage bias, while codons with value lower than 1.0 has relative negative codon usage bias. As RSCU values of some codons are nearly equal to 1.0, it means that these codons are chosen equally and randomly. The index GC3s means the fraction of the nucleotides G+C at the synonymous third codon position, excluding Met, Trp, and the termination codons. The ENC, as the best estimator of absolute synonymous codon usage bias , was calculated for the quantification of the codon usage bias of each ORF . The predicted values of ENC were calculated as ENC = 2 + s + 29 where s represents the given G+C 3 % value. The values of ENC can also be obtained by EMBOSS CHIPS program .",
"The predicted values of ENC were calculated as ENC = 2 + s + 29 where s represents the given G+C 3 % value. The values of ENC can also be obtained by EMBOSS CHIPS program . Analyses were conducted with the Nei-Gojobori model , involving 30 nucleotide sequences. All positions containing gaps and missing data were eliminated. The values of dn, ds and ω dn/ds were calculated in MEGA4.0 . Multivariate statistical analysis can be used to explore the relationships between variables and samples. In this study, correspondence analysis was used to investigate the major trend in codon usage variation among ORFs.",
"Multivariate statistical analysis can be used to explore the relationships between variables and samples. In this study, correspondence analysis was used to investigate the major trend in codon usage variation among ORFs. In this study, the complete coding region of each ORF was represented as a 59 dimensional vector, and each dimension corresponds to the RSCU value of one sense codon excluding Met, Trp, and the termination codons . Correlation analysis was used to identify the relationship between nucleotide composition and synonymous codon usage pattern . This analysis was implemented based on the Spearman's rank correlation analysis way. All statistical processes were carried out by with statistical software SPSS 17.0 for windows.",
"This analysis was implemented based on the Spearman's rank correlation analysis way. All statistical processes were carried out by with statistical software SPSS 17.0 for windows. The values of nucleotide contents in complete coding region of all 30 RHDV genomes were analyzed and listed in Table 2 and Table 3 . Evidently, C+G % content of the ORF1 fluctuated from 50.889 to 51.557 with a mean value of 51.14557, and C+G % content of the ORF2 were ranged from 35.593 to 40.113 with a mean value of 37.6624, which were indicating that nucleotides A and U were the major elements of ORF2 against ORF1. Comparing the values of A 3 %, U 3 %, C 3 % and G 3 %, it is clear that C 3 % was distinctly high and A 3 % was the lowest of all in ORF1 of RHDV, while U 3 % was distinctly high and C 3 % was the lowest of all in ORF2 of Table 2 Identified nucleotide contents in complete coding region length > 250 bps in the ORF1 of RHDV 30 isolates genome Table 4 . Most preferentially used codons in ORF1 were C-ended or G-ended codons except Ala, Pro and Ser, however, A-ended or G-ended codons were preferred as the content of ORF2.",
"Comparing the values of A 3 %, U 3 %, C 3 % and G 3 %, it is clear that C 3 % was distinctly high and A 3 % was the lowest of all in ORF1 of RHDV, while U 3 % was distinctly high and C 3 % was the lowest of all in ORF2 of Table 2 Identified nucleotide contents in complete coding region length > 250 bps in the ORF1 of RHDV 30 isolates genome Table 4 . Most preferentially used codons in ORF1 were C-ended or G-ended codons except Ala, Pro and Ser, however, A-ended or G-ended codons were preferred as the content of ORF2. In addition, the dn, ds and ω dN/dS values of ORF1 were separately 0.014, 0.338 and 0.041, and the values of ORF2 were 0.034, 0.103 and 0.034, respectively. The ω values of two ORFs in RHDV genome are generally low, indicating that the RHDV whole genome is subject to relatively strong selective constraints. COA was used to investigate the major trend in codon usage variation between two ORFs of all 30 RHDV selected for this study. After COA for RHDV Genome, one major trend in the first axis f' 1 which accounted for 42.967% of the total variation, and another major trend in the second axis f' 2 which accounted for 3.632% of the total variation.",
"COA was used to investigate the major trend in codon usage variation between two ORFs of all 30 RHDV selected for this study. After COA for RHDV Genome, one major trend in the first axis f' 1 which accounted for 42.967% of the total variation, and another major trend in the second axis f' 2 which accounted for 3.632% of the total variation. The coordinate of the complete coding region of each ORF was plotted in Figure 1 defining by the first and second principal axes. It is clear that coordinate of each ORF is relatively isolated. Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group marked as Group 1 is To estimate whether the evolution of RHDV genome on codon usage was regulated by mutation pressure or natural selection, the A%, U%, C%, G% and C+G % were compared with A 3 %, U 3 %, C 3 %, G 3 % and C 3 +G 3 %, respectively Table 5 . There is a complex correlation among nucleotide compositions.",
"Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group marked as Group 1 is To estimate whether the evolution of RHDV genome on codon usage was regulated by mutation pressure or natural selection, the A%, U%, C%, G% and C+G % were compared with A 3 %, U 3 %, C 3 %, G 3 % and C 3 +G 3 %, respectively Table 5 . There is a complex correlation among nucleotide compositions. In detail, A 3 %, U 3 %, C 3 % and G 3 % have a significant negative correlation with G%, C%, U% and A% and positive correlation with A%, U%, C% and G%, respectively. It suggests that nucleotide constraint may influence synonymous codon usage patterns. However, A 3 % has non-correlation with U% and C%, and U 3 % has noncorrelation with A% and G%, respectively, which haven't indicated any peculiarity about synonymous codon usage. Furthermore, C 3 % and G 3 % have non-correlation with A%, G% and U%, C%, respectively, indicating these data don't reflect the true feature of synonymous codon usage as well.",
"However, A 3 % has non-correlation with U% and C%, and U 3 % has noncorrelation with A% and G%, respectively, which haven't indicated any peculiarity about synonymous codon usage. Furthermore, C 3 % and G 3 % have non-correlation with A%, G% and U%, C%, respectively, indicating these data don't reflect the true feature of synonymous codon usage as well. Therefore, linear regression analysis was implemented to analyze the correlation between synonymous codon usage bias and nucleotide compositions. Details of correlation analysis between the first two principle axes f' 1 and f' 2 of each RHDV genome in COA and nucleotide contents were listed in Table 6 . In surprise, only f2 values are closely related to base nucleotide A and G content on the third codon position only, suggesting that nucleotide A and G is a factor influencing the synonymous codon usage pattern of RHDV genome. However, f' 1 value has non-correlation with base nucleotide contents on the third codon position; it is observably suggest that codon usage patterns in RHDV were probably influenced by other factors, such as the second structure of viral genome and limits of host.",
"In surprise, only f2 values are closely related to base nucleotide A and G content on the third codon position only, suggesting that nucleotide A and G is a factor influencing the synonymous codon usage pattern of RHDV genome. However, f' 1 value has non-correlation with base nucleotide contents on the third codon position; it is observably suggest that codon usage patterns in RHDV were probably influenced by other factors, such as the second structure of viral genome and limits of host. In spite of that, compositional constraint is a factor shaping the pattern of synonymous codon usage in RHDV genome. Figure 1 A plot of value of the first and second axis of RHDV genome in COA. The first axis f' 1 accounts for 42.967% of the total variation, and the second axis f' 2 accounts for 3.632% of the total variation. Table 5 Summary of correlation analysis between the A, U, C, G contents and A 3 , U 3 , C 3 , G 3 contents in all selected samples There have been more and more features that are unique to RHDV within the family Caliciviridae, including its single host tropism, its genome and its VP10 as a structural protein with unknown function.",
"The first axis f' 1 accounts for 42.967% of the total variation, and the second axis f' 2 accounts for 3.632% of the total variation. Table 5 Summary of correlation analysis between the A, U, C, G contents and A 3 , U 3 , C 3 , G 3 contents in all selected samples There have been more and more features that are unique to RHDV within the family Caliciviridae, including its single host tropism, its genome and its VP10 as a structural protein with unknown function. After we analyzed synonymous codon usage in RHDV Table 2 , we obtained several conclusions and conjectures as followed. 4.1 Mutational bias as a main factor leading to synonymous codon usage variation ENC-plot, as a general strategy, was utilized to investigate patterns of synonymous codon usage. The ENC-plots of ORFs constrained only by a C 3 +G 3 composition will lie on or just below the curve of the predicted values . ENC values of RHDV genomes were plotted against its corresponding C 3 +G 3 %.",
"The ENC-plots of ORFs constrained only by a C 3 +G 3 composition will lie on or just below the curve of the predicted values . ENC values of RHDV genomes were plotted against its corresponding C 3 +G 3 %. All of the spots lie below the curve of the predicted values, as shown in Figure 2 , suggesting that the codon usage bias in all these 30 RHDV genomes is principally influenced by the mutational bias. As we know, the efficiency of gene expression is influenced by regulator sequences or elements and codon usage bias. It reported that the RNA sequence of the 3terminal 84 nucleotides of ORF1were found to be crucial for VP10 expression instead of the encoded peptide. VP10 coding by ORF2 has been reported as a low expressive structural protein against VP60 coding by ORF1 .",
"It reported that the RNA sequence of the 3terminal 84 nucleotides of ORF1were found to be crucial for VP10 expression instead of the encoded peptide. VP10 coding by ORF2 has been reported as a low expressive structural protein against VP60 coding by ORF1 . And its efficiency of translation is only 20% of VP60. According to results showed by Table 4 , it revealed the differences in codon usage patterns of two ORFs, which is a possible factor reducing the expression of VP10. Although VP10 encoded by ORF2, as a minor structural protein with unknown functions, has been described by LIU as a nonessential protein for virus infectivity, the ω Figure 2 Effective number of codons used in each ORF plotted against the GC3s. The continuous curve plots the relationship between GC3s and ENC in the absence of selection.",
"Although VP10 encoded by ORF2, as a minor structural protein with unknown functions, has been described by LIU as a nonessential protein for virus infectivity, the ω Figure 2 Effective number of codons used in each ORF plotted against the GC3s. The continuous curve plots the relationship between GC3s and ENC in the absence of selection. All of spots lie below the expected curve. value of ORF2 suggests VP10 plays an important role in the certain stage of whole RHDV lifecycle. After combining with low expression and ω value of VP10, we conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. This mechanism has been confirmed in various positive-chain RNA viruses, including coxsackievirus, dengue virus, equine arterivirus, footand-mouth disease virus, hepatitis C virus, poliovirus, rhinovirus, and severe acute respiratory syndrome , although the details remain elusive.",
"After combining with low expression and ω value of VP10, we conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. This mechanism has been confirmed in various positive-chain RNA viruses, including coxsackievirus, dengue virus, equine arterivirus, footand-mouth disease virus, hepatitis C virus, poliovirus, rhinovirus, and severe acute respiratory syndrome , although the details remain elusive. As preceding description, ENC reflects the evolution of codon usage variation and nucleotide composition to some degree. After the correlation analysis of ENC values between ORF1 and ORF2 Table 7 , the related coefficient of ENC values of two ORFs is 0.230, and p value is 0.222 more than 0.05. These data revealed that no correlation existed in ENC values of two ORFs, indicating that codon usage patterns and evolution of two ORFs are separated each other. Further, this information maybe helps us well understand why RSCU and ENC between two ORFs are quite different.",
"These data revealed that no correlation existed in ENC values of two ORFs, indicating that codon usage patterns and evolution of two ORFs are separated each other. Further, this information maybe helps us well understand why RSCU and ENC between two ORFs are quite different. Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group marked as Group 1 is positive value and the other one marked as Group 2 is negative value. And all of those strains isolated before 2000 belonged to Group 2, including Italy-90, RHDV-V351, RHDV-FRG, BS89, RHDV-SD and M67473.1. Although RHDV has been reported as only one type, this may be a reference on dividing into two genotypes. In this report, we firstly analyzed its genome and two open reading frameworks ORFs from this aspect of codon usage bias.",
"Although RHDV has been reported as only one type, this may be a reference on dividing into two genotypes. In this report, we firstly analyzed its genome and two open reading frameworks ORFs from this aspect of codon usage bias. Our researches indicated that mutation pressure rather than natural is the most important determinant in RHDV with high codon bias, and the codon usage bias is nearly contrary between ORF1 and ORF2, which is maybe one of factors regulating the expression of VP60 encoding by ORF1 and VP10 encoding by ORF2 . Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV.",
"We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV. All the results will guide the next researches on the RHDV as a reference."
] | 1,567 | 566 |
What is the main cause of death in the neonatal period of calves?
|
Calf septicemia
|
[
"AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .",
"Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.",
"CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .",
"As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .",
"On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .",
"There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .",
"Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .",
"This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.",
"The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .",
"Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.",
"The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .",
"The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.",
"The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .",
"The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.",
"As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .",
"These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .",
"The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .",
"reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.",
"Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.",
"EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript."
] | 1,560 | 2,129 |
Where was hepcidin first discovered?
|
human urine
|
[
"AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .",
"Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.",
"CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .",
"As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .",
"On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .",
"There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .",
"Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .",
"This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.",
"The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .",
"Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.",
"The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .",
"The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.",
"The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .",
"The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.",
"As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .",
"These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .",
"The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .",
"reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.",
"Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.",
"EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript."
] | 1,560 | 2,131 |
What is hepcidin?
|
low molecular weight, antimicrobial peptide hormone
|
[
"AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .",
"Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.",
"CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .",
"As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .",
"On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .",
"There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .",
"Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .",
"This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.",
"The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .",
"Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.",
"The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .",
"The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.",
"The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .",
"The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.",
"As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .",
"These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .",
"The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .",
"reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.",
"Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.",
"EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript."
] | 1,560 | 2,130 |
What organ produces hepcidin?
|
liver
|
[
"AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .",
"Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.",
"CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .",
"As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .",
"On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .",
"There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .",
"Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .",
"This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.",
"The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .",
"Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.",
"The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .",
"The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.",
"The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .",
"The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.",
"As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .",
"These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .",
"The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .",
"reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.",
"Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.",
"EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript."
] | 1,560 | 2,132 |
What stimulates the release of hepcidin?
|
inflammatory reactions and high Fe concentrations
|
[
"AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .",
"Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.",
"CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .",
"As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .",
"On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .",
"There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .",
"Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .",
"This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.",
"The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .",
"Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.",
"The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .",
"The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.",
"The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .",
"The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.",
"As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .",
"These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .",
"The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .",
"reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.",
"Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.",
"EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript."
] | 1,560 | 2,133 |
What element does hepcidin play a roles in regulating during metabolism?
|
Fe
|
[
"AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .",
"Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.",
"CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .",
"As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .",
"On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .",
"There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .",
"Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .",
"This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.",
"The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .",
"Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.",
"The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .",
"The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.",
"The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .",
"The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.",
"As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .",
"These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .",
"The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .",
"reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.",
"Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.",
"EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript."
] | 1,560 | 2,134 |
Is hepcidin toxic?
|
potentially toxic
|
[
"AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .",
"Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.",
"CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .",
"As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .",
"On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .",
"There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .",
"Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .",
"This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.",
"The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .",
"Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.",
"The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .",
"The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.",
"The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .",
"The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.",
"As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .",
"These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .",
"The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .",
"reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.",
"Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.",
"EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript."
] | 1,560 | 2,135 |
Why is iron critical to bacteria?
|
bacteria utilize Fe for survival, growth and proliferation
|
[
"AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .",
"Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.",
"CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .",
"As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .",
"On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .",
"There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .",
"Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .",
"This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.",
"The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .",
"Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.",
"The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .",
"The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.",
"The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .",
"The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.",
"As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .",
"These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .",
"The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .",
"reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.",
"Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.",
"EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript."
] | 1,560 | 2,136 |
How does hepcidin work in the duodenum?
|
control of excessive Fe absorption
|
[
"AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .",
"Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.",
"CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .",
"As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .",
"On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .",
"There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .",
"Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .",
"This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.",
"The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .",
"Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.",
"The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .",
"The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.",
"The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .",
"The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.",
"As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .",
"These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .",
"The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .",
"reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.",
"Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.",
"EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript."
] | 1,560 | 2,137 |
How does hepcidin affect macrophages?
|
regulation of Fe release
|
[
"AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .",
"Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.",
"CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .",
"As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .",
"On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .",
"There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .",
"Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .",
"This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.",
"The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .",
"Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.",
"The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .",
"The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.",
"The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .",
"The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.",
"As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .",
"These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .",
"The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .",
"reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.",
"Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.",
"EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript."
] | 1,560 | 2,138 |
What leads to oxidative stress in the body?
|
production of ROS
|
[
"AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .",
"Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.",
"CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .",
"As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .",
"On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .",
"There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .",
"Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .",
"This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.",
"The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .",
"Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.",
"The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .",
"The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.",
"The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .",
"The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.",
"As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .",
"These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .",
"The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .",
"reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.",
"Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.",
"EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript."
] | 1,560 | 2,139 |
What parameter is used to measure antioxidant levels?
|
superoxide dismutase
|
[
"AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .",
"Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.",
"CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .",
"As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .",
"On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .",
"There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .",
"Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .",
"This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.",
"The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .",
"Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.",
"The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .",
"The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.",
"The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .",
"The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.",
"As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .",
"These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .",
"The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .",
"reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.",
"Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.",
"EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript."
] | 1,560 | 2,140 |
What is Acute hemorrhagic encephalomyelitis?
|
a rare form of acute disseminated encephalomyelitis
|
[
"BACKGROUND: Acute hemorrhagic encephalomyelitis AHEM is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein RANBP2 gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone IVMP in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease SCD presented an acquired demyelinating syndrome ADS with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma.",
"She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins IVIG . She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered.",
"After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant c.4993A>G, p.Lys1665Glu was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP.",
"RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system.",
"Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal . . . . Many treatment options have been used including intravenous IV steroids, intravenous immunoglobulins IVIG , and plasmapheresis . . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy . . . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle.",
". . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy ANE which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children . .",
"It could be triggered by a viral infection in previously healthy children . . We report a new case of AHEM associated to a Ran Binding Protein RANBP -2 variant and responsive to combined craniectomy, intravenous methylprednisolone IVMP , and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease SCD . A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia day 0 D0 : refers to the start of the diplopia 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 .",
"She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 . Brain MR angiography MRA showed a normal appearing circle of Willis. The cerebrospinal fluid CSF obtained by lumber puncture was normal WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands . The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area.",
"The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h.",
"Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP 30 mg/kg/day followed by 9 days of oral prednisone 1 mg/kg/day . At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness D30 with normal alertness. She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir.",
"She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine D30 she deteriorated with vomiting and reduced level of consciousness Glasgow Coma Scale dropped from 15 to 8 over 30 min . Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F .",
"Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis.",
"She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces.",
"She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere Figures 2G-I . She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 .",
"She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 . Prednisolone was tapered course over 6 weeks. At discharge D71 , she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma.",
"Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR HSV1/2 were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative.",
"CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens SSA-RO, SSB-LA, smith, RNP were negative. Serum autoimmune antibodies panel NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange.",
"EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu .",
"Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu . This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease 6/117,118 alleles . Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD .",
"Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD . Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . .",
"Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . . SCD patients are prone to ischemic or hemorrhagic strokes . . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . .",
"Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients . . . . . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury.",
"However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient . . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia.",
". Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis . . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . .",
"Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes . . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context . .",
"Other patients with AHEM have been described in the SCD context . . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy . . . . . . . . . . Autosomal dominant mutations with incomplete penetrance in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy . . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . .",
"Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid . It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy .",
"It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy . making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells . . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication.",
"This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation .. This work was supported by grants from Heart and Stroke Foundation of Canada grant number: G-14-0005756 , and Foundation of Stars."
] | 1,561 | 3,032 |
What are the salient findings in Acute hemorrhagic encephalomyelitis?
|
fulminant encephalopathy with hemorrhagic necrosis
|
[
"BACKGROUND: Acute hemorrhagic encephalomyelitis AHEM is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein RANBP2 gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone IVMP in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease SCD presented an acquired demyelinating syndrome ADS with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma.",
"She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins IVIG . She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered.",
"After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant c.4993A>G, p.Lys1665Glu was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP.",
"RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system.",
"Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal . . . . Many treatment options have been used including intravenous IV steroids, intravenous immunoglobulins IVIG , and plasmapheresis . . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy . . . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle.",
". . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy ANE which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children . .",
"It could be triggered by a viral infection in previously healthy children . . We report a new case of AHEM associated to a Ran Binding Protein RANBP -2 variant and responsive to combined craniectomy, intravenous methylprednisolone IVMP , and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease SCD . A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia day 0 D0 : refers to the start of the diplopia 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 .",
"She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 . Brain MR angiography MRA showed a normal appearing circle of Willis. The cerebrospinal fluid CSF obtained by lumber puncture was normal WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands . The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area.",
"The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h.",
"Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP 30 mg/kg/day followed by 9 days of oral prednisone 1 mg/kg/day . At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness D30 with normal alertness. She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir.",
"She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine D30 she deteriorated with vomiting and reduced level of consciousness Glasgow Coma Scale dropped from 15 to 8 over 30 min . Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F .",
"Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis.",
"She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces.",
"She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere Figures 2G-I . She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 .",
"She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 . Prednisolone was tapered course over 6 weeks. At discharge D71 , she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma.",
"Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR HSV1/2 were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative.",
"CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens SSA-RO, SSB-LA, smith, RNP were negative. Serum autoimmune antibodies panel NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange.",
"EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu .",
"Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu . This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease 6/117,118 alleles . Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD .",
"Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD . Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . .",
"Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . . SCD patients are prone to ischemic or hemorrhagic strokes . . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . .",
"Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients . . . . . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury.",
"However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient . . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia.",
". Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis . . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . .",
"Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes . . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context . .",
"Other patients with AHEM have been described in the SCD context . . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy . . . . . . . . . . Autosomal dominant mutations with incomplete penetrance in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy . . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . .",
"Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid . It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy .",
"It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy . making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells . . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication.",
"This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation .. This work was supported by grants from Heart and Stroke Foundation of Canada grant number: G-14-0005756 , and Foundation of Stars."
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