Hyaluronan in mesenchymal stromal cell lineage differentiation from human pluripotent stem cells: application in serum free culture.

BACKGROUND: Hyaluronan (HA) is an extracellular glycosaminoglycan polysaccharide with widespread roles throughout development and in healthy and neoplastic tissues. In pluripotent stem cell culture it can support both stem cell renewal and differentiation. However, responses to HA in culture are influenced by interaction with a range of cognate factors and receptors including components of blood serum supplements, which alter results. These may contribute to variation in cell batch production yield and phenotype as well as heighten the risks of adventitious pathogen transmission in the course of cell processing for therapeutic applications. MAIN: Here we characterise differentiation of a human embryo/pluripotent stem cell derived Mesenchymal Stromal Cell (hESC/PSC-MSC)-like cell population by culture on a planar surface coated with HA in serum-free media qualified for cell production for therapy. Resulting cells met minimum criteria of the International Society for Cellular Therapy for identification as MSC by expression of. CD90, CD73, CD105, and lack of expression for CD34, CD45, CD14 and HLA-II. They were positive for other MSC associated markers (i.e.CD166, CD56, CD44, HLA 1-A) whilst negative for others (e.g. CD271, CD71, CD146). In vitro co-culture assessment of MSC associated functionality confirmed support of growth of hematopoietic progenitors and inhibition of mitogen activated proliferation of lymphocytes from umbilical cord and adult peripheral blood mononuclear cells, respectively. Co-culture with immortalized THP-1 monocyte derived macrophages (Mɸ) concurrently stimulated with lipopolysaccharide as a pro-inflammatory stimulus, resulted in a dose dependent increase in pro-inflammatory IL6 but negligible effect on TNFα. To further investigate these functionalities, a bulk cell RNA sequence comparison with adult human bone marrow derived MSC and hESC substantiated a distinctive genetic signature more proximate to the former. CONCLUSION: Cultivation of human pluripotent stem cells on a planar substrate of HA in serum-free culture media systems is sufficient to yield a distinctive developmental mesenchymal stromal cell lineage with potential to modify the function of haematopoietic lineages in therapeutic applications.

Transient increase in atherosclerotic plaque macrophage content following Streptococcus pneumoniae pneumonia in ApoE-deficient mice.

Introduction: Despite epidemiological associations between community acquired pneumonia (CAP) and myocardial infarction, mechanisms that modify cardiovascular disease during CAP are not well defined. In particular, largely due to a lack of relevant experimental models, the effect of pneumonia on atherosclerotic plaques is unclear. We describe the development of a murine model of the commonest cause of CAP, Streptococcus pneumoniae pneumonia, on a background of established atherosclerosis. We go on to use our model to investigate the effects of pneumococcal pneumonia on atherosclerosis. Methods: C57BL/6J and ApoE-/- mice were fed a high fat diet to promote atherosclerotic plaque formation. Mice were then infected with a range of S. pneumoniae serotypes (1, 4 or 14) with the aim of establishing a model to study atherosclerotic plaque evolution after pneumonia and bacteremia. Laser capture microdissection of plaque macrophages enabled transcriptomic analysis. Results: Intratracheal instillation of S. pneumoniae in mice fed a cholate containing diet resulted in low survival rates following infection, suggestive of increased susceptibility to severe infection. Optimization steps resulted in a final model of male ApoE-/- mice fed a Western diet then infected by intranasal instillation of serotype 4 (TIGR4) S. pneumoniae followed by antibiotic administration. This protocol resulted in high rates of bacteremia (88.9%) and survival (88.5%). Pneumonia resulted in increased aortic sinus plaque macrophage content 2 weeks post pneumonia but not at 8 weeks, and no difference in plaque burden or other plaque vulnerability markers were found at either time point. Microarray and qPCR analysis of plaque macrophages identified downregulation of two E3 ubiquitin ligases, Huwe1 and Itch, following pneumonia. Treatment with atorvastatin failed to alter plaque macrophage content or other plaque features. Discussion: Without antibiotics, ApoE-/- mice fed a high fat diet were highly susceptible to mortality following S. pneumoniae infection. The major infection associated change in plaque morphology was an early increase in plaque macrophages. Our results also hint at a role for the ubiquitin proteasome system in the response to pneumococcal infection in the plaque microenvironment.

Upper respiratory tract mucosal immunity for SARS-CoV-2 vaccines.

SARS-CoV-2 vaccination significantly reduces morbidity and mortality, but has less impact on viral transmission rates, thus aiding viral evolution, and the longevity of vaccine-induced immunity rapidly declines. Immune responses in respiratory tract mucosal tissues are crucial for early control of infection, and can generate long-term antigen-specific protection with prompt recall responses. However, currently approved SARS-CoV-2 vaccines are not amenable to adequate respiratory mucosal delivery, particularly in the upper airways, which could account for the high vaccine breakthrough infection rates and limited duration of vaccine-mediated protection. In view of these drawbacks, we outline a strategy that has the potential to enhance both the efficacy and durability of existing SARS-CoV-2 vaccines, by inducing robust memory responses in the upper respiratory tract (URT) mucosa.

NRF2 Activation Reprograms Defects in Oxidative Metabolism to Restore Macrophage Function in Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is a disease characterized by persistent airway inflammation and disordered macrophage function. The extent to which alterations in macrophage bioenergetics contribute to impaired antioxidant responses and disease pathogenesis has yet to be fully delineated. Objectives: Through the study of COPD alveolar macrophages (AMs) and peripheral monocyte-derived macrophages (MDMs), we sought to establish if intrinsic defects in core metabolic processes drive macrophage dysfunction and redox imbalance. Methods: AMs and MDMs from donors with COPD and healthy donors underwent functional, metabolic, and transcriptional profiling. Measurements and Main Results: We observed that AMs and MDMs from donors with COPD display a critical depletion in glycolytic- and mitochondrial respiration-derived energy reserves and an overreliance on glycolysis as a source for ATP, resulting in reduced energy status. Defects in oxidative metabolism extend to an impaired redox balance associated with defective expression of the NADPH-generating enzyme, ME1 (malic enzyme 1), a known target of the antioxidant transcription factor NRF2 (nuclear factor erythroid 2-related factor 2). Consequently, selective activation of NRF2 resets the COPD transcriptome, resulting in increased generation of TCA cycle intermediaries, improved energetic status, favorable redox balance, and recovery of macrophage function. Conclusions: In COPD, an inherent loss of metabolic plasticity leads to metabolic exhaustion and reduced redox capacity, which can be rescued by activation of the NRF2 pathway. Targeting these defects, via NRF2 augmentation, may therefore present an attractive therapeutic strategy for the treatment of the aberrant airway inflammation described in COPD.

An Inhaled Galectin-3 Inhibitor in COVID-19 Pneumonitis: A Phase Ib/IIa Randomized Controlled Clinical Trial (DEFINE).

Rationale: High circulating galectin-3 is associated with poor outcomes in patients with coronavirus disease (COVID-19). We hypothesized that GB0139, a potent inhaled thiodigalactoside galectin-3 inhibitor with antiinflammatory and antifibrotic actions, would be safely and effectively delivered in COVID-19 pneumonitis. Objectives: Primary outcomes were safety and tolerability of inhaled GB0139 as an add-on therapy for patients hospitalized with COVID-19 pneumonitis. Methods: We present the findings of two arms of a phase Ib/IIa randomized controlled platform trial in hospitalized patients with confirmed COVID-19 pneumonitis. Patients received standard of care (SoC) or SoC plus 10 mg inhaled GB0139 twice daily for 48 hours, then once daily for up to 14 days or discharge. Measurements and Main Results: Data are reported from 41 patients, 20 of which were assigned randomly to receive GB0139. Primary outcomes: the GB0139 group experienced no treatment-related serious adverse events. Incidences of adverse events were similar between treatment arms (40 with GB0139 + SoC vs. 35 with SoC). Secondary outcomes: plasma GB0139 was measurable in all patients after inhaled exposure and demonstrated target engagement with decreased circulating galectin (overall treatment effect post-hoc analysis of covariance [ANCOVA] over days 2-7; P = 0.0099 vs. SoC). Plasma biomarkers associated with inflammation, fibrosis, coagulopathy, and major organ function were evaluated. Conclusions: In COVID-19 pneumonitis, inhaled GB0139 was well-tolerated and achieved clinically relevant plasma concentrations with target engagement. The data support larger clinical trials to determine clinical efficacy. Clinical trial registered with ClinicalTrials.gov (NCT04473053) and EudraCT (2020-002230-32).

Systematic comparison of ranking aggregation methods for gene lists in experimental results.

MOTIVATION: A common experimental output in biomedical science is a list of genes implicated in a given biological process or disease. The gene lists resulting from a group of studies answering the same, or similar, questions can be combined by ranking aggregation methods to find a consensus or a more reliable answer. Evaluating a ranking aggregation method on a specific type of data before using it is required to support the reliability since the property of a dataset can influence the performance of an algorithm. Such evaluation on gene lists is usually based on a simulated database because of the lack of a known truth for real data. However, simulated datasets tend to be too small compared to experimental data and neglect key features, including heterogeneity of quality, relevance and the inclusion of unranked lists. RESULTS: In this study, a group of existing methods and their variations that are suitable for meta-analysis of gene lists are compared using simulated and real data. Simulated data were used to explore the performance of the aggregation methods as a function of emulating the common scenarios of real genomic data, with various heterogeneity of quality, noise level and a mix of unranked and ranked data using 20 000 possible entities. In addition to the evaluation with simulated data, a comparison using real genomic data on the SARS-CoV-2 virus, cancer (non-small cell lung cancer) and bacteria (macrophage apoptosis) was performed. We summarize the results of our evaluation in a simple flowchart to select a ranking aggregation method, and in an automated implementation using the meta-analysis by information content algorithm to infer heterogeneity of data quality across input datasets. AVAILABILITY AND IMPLEMENTATION: The code for simulated data generation and running edited version of algorithms: https://github.com/baillielab/comparison_of_RA_methods. Code to perform an optimal selection of methods based on the results of this review, using the MAIC algorithm to infer the characteristics of an input dataset, can be downloaded here: https://github.com/baillielab/maic. An online service for running MAIC: https://baillielab.net/maic. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Introducing pulse oximetry for outpatient management of childhood pneumonia: An implementation research adopting a district implementation model in selected rural facilities in Bangladesh.

Background: Pulse oximetry has potential for identifying hypoxaemic pneumonia and substantially reducing under-five deaths in low- and middle-income countries (LMICs) setting. However, there are few examples of introducing pulse oximetry in resource-constrained paediatric outpatient settings, such as Integrated Management of Childhood Illness (IMCI) services. Methods: The National IMCI-programme of Bangladesh designed and developed a district implementation model for introducing pulse oximetry in routine IMCI services through stakeholder engagement and demonstrated the model in Kushtia district adopting a health system strengthening approach. Between December 2020 and June 2021, two rounds of assessment were conducted based on WHO's implementation research framework and outcome variables, involving 22 IMCI service-providers and 1680 children presenting with cough/difficulty-in-breathing in 12 health facilities. The data collection procedures included structured-observations, re-assessments, interviews, and data-extraction by trained study personnel. Findings: We observed that IMCI service-providers conducted pulse oximetry assessments on all eligible children in routine outpatient settings, of which 99% of assessments were successful; 85% (95% CI 83,87) in one attempt, and 69% (95% CI 67,71) within one minute. The adherence to standard operating procedure related to pulse oximetry was 92% (95% CI 91,93), and agreement regarding identifying hypoxaemia was 97% (95% CI 96,98). The median performance-time was 36 seconds (IQR 20,75), which was longer among younger children (2-11 months: 44s, IQR 22,78; 12-59 months: 30s, IQR 18,53, p < 0.01) and among those classified as pneumonia/severe-pneumonia than as no-pneumonia (41s, IQR 22,70; 32s, IQR 20,62, p < 0.01). We observed improvements in almost all indicators in round-2. IMCI service-providers and caregivers showed positive attitudes towards using this novel technology for assessing their children. Interpretation: This implementation research study suggested the adoption, feasibility, fidelity, appropriateness, acceptability, and sustainability of pulse oximetry introduction in routine IMCI services in resource-poor settings. The learning may inform the evidence-based scale-up of pulse oximetry linked with an oxygen delivery system in Bangladesh and other LMICs. Funding: This research was funded by the UK National Institute for Health Research (NIHR) (Global Health Research Unit on Respiratory Health (RESPIRE); 16/136/109) using UK aid from the UK Government to support global health research.

Success and time implications of SpO2 measurement through pulse oximetry among hospitalised children in rural Bangladesh: Variability by various device-, provider- and patient-related factors.

Background: Hypoxaemia is one of the strongest predictors of mortality among children with pneumonia. It can be identified through pulse oximetry instantaneously, which is a non-invasive procedure but can be influenced by factors related to the specific measuring device, health provider and patient. Following WHO's global recommendation in 2014, Bangladesh decided to introduce pulse oximetry in paediatric outpatient services, ie, the Integrated Management of Childhood Illness (IMCI) services in 2019. A national committee updated the existing IMCI implementation package and decided to test it by assessing the pulse oximetry performance of different types of assessors in real-life inpatient settings. Methods: We adopted an observational design and conducted a technology assessment among children admitted to a rural district hospital. Eleven nurses and seven paramedics received one-day training on pulse oximetry as assessors. Each assessor performed at least 30 pulse oximetry measurements on children with two types of handheld devices. The primary outcome of interest was obtaining a successful measurement of SpO2, defined as observing a stable (±1%) reading for at least 10 seconds. Performance time, ie, time taken to obtain a successful measurement of SpO2 was considered the secondary outcome of interest. In addition, we used Generalized Estimating Equation to assess the effect of different factors on the pulse oximetry performance. Results: The assessors obtained successful measurements of SpO2 in all attempts (n = 1478) except one. The median time taken was 30 (interquartile range (IQR) = 22-42) seconds, and within 60 seconds, 92% of attempts were successful. The odds of obtaining a successful measurement within 60 seconds were 7.3 (95% confidence interval (CI) = 3.7-14.2) times higher with a Masimo device than a Lifebox device. Similarly, assessors aged >25 years were 4.8 (95% CI = 1.2, 18.6) times more likely to obtain a successful measurement within 60 seconds. The odds of obtaining a successful measurement was 2.6 (95% CI = 1.6, 4.2) times higher among children aged 12-59 months compared to 2-11 months. Conclusions: Our study indicated that assessors could achieve the necessary skills to perform pulse oximetry successfully in real-life inpatient settings through a short training module, with some effect of device-, provider- and patient-related factors. The National IMCI Programme of Bangladesh can use these findings for finalising the national IMCI training modules and implementation package incorporating the recommendation of using pulse oximetry for childhood pneumonia assessment.

Hypoxia shapes the immune landscape in lung injury and promotes the persistence of inflammation.

Hypoxemia is a defining feature of acute respiratory distress syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, yet the resulting tissue hypoxia, and its impact on immune responses, is often neglected. In the present study, we have shown that ARDS patients were hypoxemic and monocytopenic within the first 48 h of ventilation. Monocytopenia was also observed in mouse models of hypoxic acute lung injury, in which hypoxemia drove the suppression of type I interferon signaling in the bone marrow. This impaired monopoiesis resulted in reduced accumulation of monocyte-derived macrophages and enhanced neutrophil-mediated inflammation in the lung. Administration of colony-stimulating factor 1 in mice with hypoxic lung injury rescued the monocytopenia, altered the phenotype of circulating monocytes, increased monocyte-derived macrophages in the lung and limited injury. Thus, tissue hypoxia altered the dynamics of the immune response to the detriment of the host and interventions to address the aberrant response offer new therapeutic strategies for ARDS.

Introducing pulse oximetry in routine IMCI services in Bangladesh: A context-driven approach to influence policy and programme through stakeholder engagement.

Background: Pneumonia is the leading cause of under-five child deaths globally and in Bangladesh. Hypoxaemia or low (<90%) oxygen concentration in the arterial blood is one of the strongest predictors of child mortality from pneumonia and other acute respiratory infections. Since 2014, the World Health Organization recommends using pulse oximetry devices in Integrated Management of Childhood Illness (IMCI) services (outpatient child health services), but it was not routinely used in most health facilities in Bangladesh until 2018. This paper describes the stakeholder engagement process embedded in an implementation research study to influence national policy and programmes to introduce pulse oximetry in routine IMCI services in Bangladesh. Methods: Based on literature review and expert consultations, we developed a conceptual framework, which guided the planning and implementation of a 4-step stakeholder engagement process. Desk review, key informant interviews, consultative workshops and onsite demonstration were the key methods to involve and engage a wide range of stakeholders. In the first step, a comprehensive desk review and key informant interviews were conducted to identify stakeholder organisations and scored them based on their power and interest levels regarding IMCI implementation in Bangladesh. In the second step, two national level, two district level and five sub-district level sensitisation workshops were organised to orient all stakeholder organisations having high power or high interest regarding the importance of using pulse oximetry for pneumonia assessment and classification. In the third step, national and district level high power-high interest stakeholder organisations were involved in developing a joint action plan for introducing pulse oximetry in routine IMCI services. In the fourth step, led by a formal working group under the leadership of the Ministry of Health, we updated the national IMCI implementation package, including all guidelines, training manuals, services registers and referral forms in English and Bangla. Subsequently, we demonstrated its use in real-life settings involving various levels of (national, district and sub-district) stakeholders and worked alongside the government leaders towards carefully resuming activities despite the COVID-19 pandemic. Results: Our engagement process contributed to the national decision to introduce pulse oximetry in routine child health services and update the national IMCI implementation package demonstrating country ownership, government leadership and multi-partner involvement, which are steppingstones towards scalability and sustainability. However, our experience clearly delineates that stakeholder engagement is a context-driven, time-consuming, resource-intensive, iterative, mercurial process that demands meticulous planning, prioritisation, inclusiveness, and adaptability. It is also influenced by the expertise, experience and positionality of the facilitating organization. Conclusions: Our experience has demonstrated the value and potential of the approach that we adopted for stakeholder engagement. However, the approach needs to be conceptualised coupled with the allocation of adequate resources and time commitment to implement it effectively.

Neutrophil HIF-1α stabilization is augmented by mitochondrial ROS produced via the glycerol 3-phosphate shuttle.

Neutrophils are predominantly glycolytic cells that derive little ATP from oxidative phosphorylation; however, they possess an extensive mitochondrial network and maintain a mitochondrial membrane potential. Although studies have shown neutrophils need their mitochondria to undergo apoptosis and regulate NETosis, the metabolic role of the respiratory chain in these highly glycolytic cells is still unclear. Recent studies have expanded on the role of reactive oxygen species (ROS) released from the mitochondria as intracellular signaling molecules. Our study shows that neutrophils can use their mitochondria to generate ROS and that mitochondrial ROS release is increased in hypoxic conditions. This is needed for the stabilization of a high level of the critical hypoxic response factor and pro-survival protein HIF-1α in hypoxia. Further, we demonstrate that neutrophils use the glycerol 3-phosphate pathway as a way of directly regulating mitochondrial function through glycolysis, specifically to maintain polarized mitochondria and produce ROS. This illustrates an additional pathway by which neutrophils can regulate HIF-1α stability and will therefore be an important consideration when looking for treatments of inflammatory conditions in which HIF-1α activation and neutrophil persistence at the site of inflammation are linked to disease severity.

The effect of live attenuated influenza vaccine on pneumococcal colonisation densities among children aged 24-59 months in The Gambia: a phase 4, open label, randomised, controlled trial.

BACKGROUND: Influenza and other respiratory viruses promote Streptococcus pneumoniae proliferation in the upper respiratory tract. We sought to investigate for what we believe is the first time, the effect of intranasal live attenuated influenza vaccine (LAIV) on nasopharyngeal S pneumoniae density in a low-income to middle-income country population with high pneumococcal carriage rates. METHODS: In an open-label, randomised, controlled trial in The Gambia, 330 healthy children aged 24-59 months were randomly assigned 2:1 to receive one trivalent LAIV dose at enrolment (day 0, intervention) or at the end of active follow-up (day 21, control). The investigator team were initially masked to block size and randomisation sequence to avoid allocation bias. Group allocation was later revealed to the investigator team. The primary outcome was PCR-quantified day 7 and 21 pneumococcal density. Asymptomatic respiratory viral infection at baseline and LAIV strain shedding were included as covariates in generalised mixed-effects models, to assess the effect of LAIV and other variables on pneumococcal densities. The study is registered at ClinicalTrials.gov, NCT02972957, and is closed to recruitment. FINDINGS: Between Feb 8 and April 12, 2017, and Jan 15 and March 28, 2018, of 343 children assessed for eligibility, 213 in the intervention group and 108 in the control group completed the study and were included in the final analysis. Although no significant differences were seen in pneumococcal carriage or density at each timepoint when comparing groups, changes from baseline were observed in the LAIV group. The baseline S pneumoniae carriage prevalence was high in both LAIV and control groups (75%) and increased by day 21 in the LAIV group (85%, p=0·0037), but not in the control group (79%, p=0·44). An increase in pneumococcal density from day 0 amounts was seen in the LAIV group at day 7 (+0·207 log10 copies per µL, SE 0·105, p=0·050) and day 21 (+0·280 log10 copies per µL, SE 0·105, p=0·0082), but not in the control group. Older age was associated with lower pneumococcal density (-0·015 log10 copies per µL, SE 0·005, p=0·0030), with the presence of asymptomatic respiratory viruses at baseline (+0·259 log10 copies per µL, SE 0·097, p=0·017), and greater LAIV shedding at day 7 (+0·380 log10 copies per µL, SE 0·167, p=0·024) associated with higher pneumococcal density. A significant increase in rhinorrhoea was reported in the LAIV group compared with the control group children during the first 7 days of the study (103 [48%] of 213, compared with 25 [23%] of 108, p<0·0001), and between day 7 and 21 (108 [51%] of 213, compared with 28 [26%] of 108, p<0·0001). INTERPRETATION: LAIV was associated with a modest increase in nasopharyngeal pneumococcal carriage and density in the 21 days following vaccination, with the increase in density lower in magnitude than previously described in the UK. This increase was accelerated when LAIV was administered in the presence of pre-existing asymptomatic respiratory viruses, suggesting that nasopharyngeal S pneumoniae proliferation is driven by cumulative mixed-viral co-infections. The effect of LAIV on pneumococcal density is probably similar to other respiratory viral infections in children. Our findings provide reassurance for the use of LAIV to expand influenza vaccine programmes in low-income to middle-income country populations with high pneumococcal carriage. FUNDING: Wellcome Trust.

The transcription factor EGR2 is indispensable for tissue-specific imprinting of alveolar macrophages in health and tissue repair.

Alveolar macrophages are the most abundant macrophages in the healthy lung where they play key roles in homeostasis and immune surveillance against airborne pathogens. Tissue-specific differentiation and survival of alveolar macrophages rely on niche-derived factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor­ß (TGF-ß). However, the nature of the downstream molecular pathways that regulate the identity and function of alveolar macrophages and their response to injury remain poorly understood. Here, we identify that the transcription factor EGR2 is an evolutionarily conserved feature of lung alveolar macrophages and show that cell-intrinsic EGR2 is indispensable for the tissue-specific identity of alveolar macrophages. Mechanistically, we show that EGR2 is driven by TGF-ß and GM-CSF in a PPAR-γ­dependent manner to control alveolar macrophage differentiation. Functionally, EGR2 was dispensable for the regulation of lipids in the airways but crucial for the effective handling of the respiratory pathogen Streptococcus pneumoniae. Last, we show that EGR2 is required for repopulation of the alveolar niche after sterile, bleomycin-induced lung injury and demonstrate that EGR2-dependent, monocyte-derived alveolar macrophages are vital for effective tissue repair after injury. Collectively, we demonstrate that EGR2 is an indispensable component of the transcriptional network controlling the identity and function of alveolar macrophages in health and disease.

Child mortality in Bangladesh - why, when, where and how? A national survey-based analysis.

BACKGROUND: Updated information on the cause of childhood mortality is essential for developing policies and designing programmes targeting the major burden of disease. There is a paucity of evidence regarding the current estimates of the cause of death in Bangladesh, which is essential for reinvigorating the current policies and reshaping existing strategies to avert preventable deaths. This paper aims to address this critical evidence gap and report the cause, timing and place of death among children under-five years of age using a nationally representative sample. METHODS: The present study was undertaken to provide updated estimates of causes of death among children under-five years of age using data from the 2017-18 round of the Bangladesh Demographic and Health Survey (BDHS). The verbal autopsy (VA) questionnaire of the 2017-18 BDHS was adapted from the standardised WHO 2016 instruments. Specially trained physicians reviewed the responses of the VA questionnaire and assigned the cause of death based on the online-2016-version of the International Classification of Diseases (ICD-10). We included 456 deaths among children under-five years of age in our analysis. Descriptive statistics were used to present the causes, timing and places of death with uncertainty ranges (UR). RESULTS: Pneumonia is the major killer (19%), accounting for approximately 24 268 (UR = 21 626-26 695) under-five deaths per-year. It is followed by birth asphyxia (16%), prematurity and low-birth-weight (11%), serious infections including sepsis (8%) causing 20 882 (UR = 18 608-22 970), 14 956 (UR = 13 327-16,452), and 10 723 (UR = 9555-11,795) deaths per-year, respectively. Drowning (8%) caused 10 441 (UR = 9304-11 485) deaths and congenital anomaly (7%) resulted in d 8748 (UR = 7795-9623) deaths per-year. Around 29% of all deaths occurred on the first day, 52% within the first week, and 66% within the first month of life. Around 70% of birth asphyxia, prematurity, and low birth weight-related deaths happen on the day of birth. Approximately 43% of pneumonia-related deaths occur in age 1-11 months, and around 51% of drowning-related deaths happen in age 12-23 months. CONCLUSIONS: Pneumonia with other serious infections, birth asphyxia, prematurity and low-birth-weight are responsible for more than half of all deaths among children under-five years of age. Strengthening the existing maternal, neonatal and child health programmes may be helpful in averting the majority of these preventable deaths. A multisectoral approach is required for the prevention of childhood deaths, especially drowning-related fatalities. Special measures need to be taken to prevent and control emerging public health challenges like birth defects and congenital anomalies.

Hypoxaemia prevalence and its adverse clinical outcomes among children hospitalised with WHO-defined severe pneumonia in Bangladesh.

BACKGROUND: With an estimated 1 million cases per year, pneumonia accounts for 15% of all under-five deaths globally, and hypoxaemia is one of the strongest predictors of mortality. Most of these deaths are preventable and occur in low- and middle-income countries. Bangladesh is among the six high burden countries with an estimated 4 million pneumonia episodes annually. There is a gap in updated evidence on the prevalence of hypoxaemia among children with severe pneumonia in high burden countries, including Bangladesh. METHODS: We conducted a secondary analysis of data obtained from icddr,b-Dhaka Hospital, a secondary level referral hospital located in Dhaka, Bangladesh. We included 2646 children aged 2-59 months admitted with WHO-defined severe pneumonia during 2014-17. The primary outcome of interest was hypoxaemia, defined as SpO2 < 90% on admission. The secondary outcome of interest was adverse clinical outcomes defined as deaths during hospital stay or referral to higher-level facilities due to clinical deterioration. RESULTS: On admission, the prevalence of hypoxaemia among children hospitalised with severe pneumonia was 40%. The odds of hypoxaemia were higher among females (adjusted Odds ratio AOR = 1.44; 95% confidence interval CI = 1.22-1.71) and those with a history of cough or difficulty in breathing for 0-48 hours before admission (AOR = 1.61; 95% CI = 1.28-2.02). Among all children with severe pneumonia, 6% died during the hospital stay, and 9% were referred to higher-level facilities due to clinical deterioration. Hypoxaemia was the strongest predictor of mortality (AOR = 11.08; 95% CI = 7.28-16.87) and referral (AOR = 5.94; 95% CI = 4.31-17) among other factors such as age, sex, history of fever and cough or difficulty in breathing, and severe acute malnutrition. Among those who survived, the median duration of hospital stay was 7 (IQR = 4-11) days in the hypoxaemic group and 6 (IQR = 4-9) days in the non-hypoxaemic group, and the difference was significant at P < 0.001. CONCLUSIONS: The high burden of hypoxaemia and its clinical outcomes call for urgent attention to promote oxygen security in low resource settings like Bangladesh. The availability of pulse oximetry for rapid identification and an effective oxygen delivery system for immediate correction should be ensured for averting many preventable deaths.

The histone methyltransferase Ezh2 restrains macrophage inflammatory responses.

Robust inflammatory responses are critical to survival following respiratory infection, with current attention focused on the clinical consequences of the Coronavirus pandemic. Epigenetic factors are increasingly recognized as important determinants of immune responses, and EZH2 is a prominent target due to the availability of highly specific and efficacious antagonists. However, very little is known about the role of EZH2 in the myeloid lineage. Here, we show EZH2 acts in macrophages to limit inflammatory responses to activation, and in neutrophils for chemotaxis. Selective genetic deletion in macrophages results in a remarkable gain in protection from infection with the prevalent lung pathogen, pneumococcus. In contrast, neutrophils lacking EZH2 showed impaired mobility in response to chemotactic signals, and resulted in increased susceptibility to pneumococcus. In summary, EZH2 shows complex, and divergent roles in different myeloid lineages, likely contributing to the earlier conflicting reports. Compounds targeting EZH2 are likely to impair mucosal immunity; however, they may prove useful for conditions driven by pulmonary neutrophil influx, such as adult respiratory distress syndrome.

Pneumolysin Is Responsible for Differential Gene Expression and Modifications in the Epigenetic Landscape of Primary Monocyte Derived Macrophages.

Epigenetic modifications regulate gene expression in the host response to a diverse range of pathogens. The extent and consequences of epigenetic modification during macrophage responses to Streptococcus pneumoniae, and the role of pneumolysin, a key Streptococcus pneumoniae virulence factor, in influencing these responses, are currently unknown. To investigate this, we infected human monocyte derived macrophages (MDMs) with Streptococcus pneumoniae and addressed whether pneumolysin altered the epigenetic landscape and the associated acute macrophage transcriptional response using a combined transcriptomic and proteomic approach. Transcriptomic analysis identified 503 genes that were differentially expressed in a pneumolysin-dependent manner in these samples. Pathway analysis highlighted the involvement of transcriptional responses to core innate responses to pneumococci including modules associated with metabolic pathways activated in response to infection, oxidative stress responses and NFκB, NOD-like receptor and TNF signalling pathways. Quantitative proteomic analysis confirmed pneumolysin-regulated protein expression, early after bacterial challenge, in representative transcriptional modules associated with innate immune responses. In parallel, quantitative mass spectrometry identified global changes in the relative abundance of histone post translational modifications (PTMs) upon pneumococcal challenge. We identified an increase in the relative abundance of H3K4me1, H4K16ac and a decrease in H3K9me2 and H3K79me2 in a PLY-dependent fashion. We confirmed that pneumolysin blunted early transcriptional responses involving TNF-α and IL-6 expression. Vorinostat, a histone deacetylase inhibitor, similarly downregulated TNF-α production, reprising the pattern observed with pneumolysin. In conclusion, widespread changes in the macrophage transcriptional response are regulated by pneumolysin and are associated with global changes in histone PTMs. Modulating histone PTMs can reverse pneumolysin-associated transcriptional changes influencing innate immune responses, suggesting that epigenetic modification by pneumolysin plays a role in dampening the innate responses to pneumococci.