Respiratory Syncytial Virus-related Community Chronic Obstructive Pulmonary Disease Exacerbations and Novel Diagnostics: A Binational Prospective Cohort Study.

Rationale: Respiratory syncytial virus (RSV) is a common global respiratory virus that is increasingly recognized as a major pathogen in frail older adults and as a cause of chronic obstructive pulmonary disease (COPD) exacerbations. There is no single test for RSV in adults that has acceptable diagnostic accuracy. Trials of RSV vaccines have recently shown excellent safety and efficacy against RSV in older adults; defining the frequency of RSV-related community infections and COPD exacerbations is important for vaccine deployment decisions. Objectives: This prospective study aimed to establish the frequency of outpatient-managed RSV-related exacerbations of COPD in two well-characterized patient cohorts using a combination of diagnostic methods. Methods: Participants were recruited at specialist clinics in London, United Kingdom, and Groningen, the Netherlands, beginning in 2017 and observed for three consecutive RSV seasons, during exacerbations, and at least twice yearly. RSV infections were detected by RT-PCR and serologic testing. Measurements and Main Results: A total of 377 patients with COPD attended 1,999 clinic visits and reported 310 exacerbations. There were 27 RSV-related exacerbations (8.7% of the total); of these, seven were detected only by PCR, 16 only by serology, and four by both methods. Increases in RSV-specific Nucleoprotein antibody were as sensitive as those in the antibody to Pre-Fusion or Post-Fusion for serodiagnosis of RSV-related exacerbations. Conclusions: RSV is associated with 8.7% of outpatient-managed COPD exacerbations in this study. Antibodies to RSV Nucleoprotein may have diagnostic value and are potentially important in a vaccinated population. The introduction of vaccines that prevent RSV is expected to benefit patients with COPD.

External Validation of the Discriminative Validity of the ReSVinet Score and Development of Simplified ReSVinet Scores in Secondary Care.

BACKGROUND: There is no consensus on how to best quantify disease severity in infants with respiratory syncytial virus (RSV) and/or bronchiolitis; this lack of a sufficiently validated score complicates the provision of clinical care and, the evaluation of trials of therapeutics and vaccines. The ReSVinet score appears to be one of the most promising; however, it is too time consuming to be incorporated into routine clinical care. We aimed to develop and externally validate simplified versions of this score. METHODS: Data from a multinational (the Netherlands, Spain, and United Kingdom) multicenter case-control study of infants with RSV were used to develop simplified versions of the ReSVinet score by conducting a grid search to determine the best combination of equally weighted parameters to maximize for the discriminative ability (measured by area under the receiver operating characteristic curve [AUROC]) across a range of outcomes (hospitalization, intensive care unit admission, ventilation requirement). Subsequently discriminative validity of the score for a range of secondary care outcomes was externally validated by secondary analysis of datasets from Rwanda and Colombia. RESULTS: Three candidate simplified scores were identified using the development dataset; they were excellent (AUROC >0.9) at discriminating for a range of outcomes, and their performance was not significantly different from the original ReSVinet score despite having fewer parameters. In the external validation datasets, the simplified scores were moderate to excellent (AUROC, 0.7-1) across a range of outcomes. In all outcomes, except in a single dataset for predicting admission to the high-dependency unit, they performed at least as well as the original ReSVinet score. CONCLUSIONS: The candidate simplified scores developed require further external validation in larger datasets, ideally from resource-limited settings before any recommendation regarding their use.

A Genome-Wide Association Study of Respiratory Syncytial Virus Infection Severity in Infants.

BACKGROUND: Respiratory syncytial virus (RSV) is a significant cause of infant morbidity and mortality worldwide. Most children experience at least one 1 RSV infection by the age of two 2 years, but not all develop severe disease. However, the understanding of genetic risk factors for severe RSV is incomplete. Consequently, we conducted a genome-wide association study of RSV severity. METHODS: Disease severity was assessed by the ReSVinet scale, in a cohort of 251 infants aged 1 week to 1 year. Genotyping data were collected from multiple European study sites as part of the RESCEU Consortium. Linear regression models were used to assess the impact of genotype on RSV severity and gene expression as measured by microarray. RESULTS: While no SNPs reached the genome-wide statistical significance threshold (P < 5 × 10-8), we identified 816 candidate SNPs with a P-value of <1 × 10-4. Functional annotation of candidate SNPs highlighted genes relevant to neutrophil trafficking and cytoskeletal functions, including LSP1 and RAB27A. Moreover, SNPs within the RAB27A locus significantly altered gene expression (false discovery rate, FDR P < .05). CONCLUSIONS: These findings may provide insights into genetic mechanisms driving severe RSV infection, offering biologically relevant information for future investigations.

A Systematic Review and Meta-analysis of Animal Studies Investigating the Relationship Between Serum Antibody, T Lymphocytes, and Respiratory Syncytial Virus Disease.

BACKGROUND: Respiratory syncytial virus (RSV) infections occur in human populations around the globe, causing disease of variable severity, disproportionately affecting infants and older adults (>65 years of age). Immune responses can be protective but also contribute to disease. Experimental studies in animals enable detailed investigation of immune responses, provide insights into clinical questions, and accelerate the development of passive and active vaccination. We aimed to review the role of antibody and T-cell responses in relation to RSV disease severity in animals. METHODS: Systematic review and meta-analysis of animal studies examining the association between T-cell responses/phenotype or antibody titers and severity of RSV disease. The PubMed, Zoological Record, and Embase databases were screened from January 1980 to May 2018 to identify animal studies of RSV infection that assessed serum antibody titer or T lymphocytes with disease severity as an outcome. Sixty-three studies were included in the final review. RESULTS: RSV-specific antibody appears to protect from disease in mice, but such an effect was less evident in bovine RSV. Strong T-cell, Th1, Th2, Th17, CD4/CD8 responses, and weak Treg responses accompany severe disease in mice. CONCLUSIONS: Murine studies suggest that measures of T-lymphocyte activity (particularly CD4 and CD8 T cells) may be predictive biomarkers of severity. Further inquiry is merited to validate these results and assess relevance as biomarkers for human disease.

Clinical and Viral Factors Associated With Disease Severity and Subsequent Wheezing in Infants With Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in infants and young children worldwide. Here we evaluated host demographic and viral factors associated with RSV disease severity in 325 RSV-infected infants under 1 year of age from 3 European countries during 2017-2020. Younger infants had a higher clinical severity (ReSViNET) score and were more likely to require hospitalization, intensive care, respiratory support, and/or mechanical ventilation than older infants (<3 months vs 3 to <6 months and 3 to <6 months vs ≥6 months). Older age (≥6 months vs <3 months), higher viral load, and RSV-A were associated with a greater probability of fever. RSV-A and RSV-B caused similar disease severity and had similar viral dynamics. Infants with a more severe RSV infection, demonstrated by having a higher ReSViNET score, fever, and requiring hospitalization and intensive care, were more likely to have developed subsequent wheezing at 1 year of age. CLINICAL TRIALS REGISTRATION: NCT03756766.

Burden of respiratory syncytial virus infection in community-dwelling older adults in Europe (RESCEU): an international prospective cohort study.

BACKGROUND: Respiratory syncytial virus (RSV) infection in older adults is recognised as an important health issue. We aimed to assess the community burden of RSV in Europe in older adults aged ≥60 years. METHODS: This international, prospective, observational cohort study is part of work by the REspiratory Syncytial virus Consortium in EUrope (RESCEU). Participants were recruited through general practitioners' (GPs) offices before two independent RSV seasons. Participants reported weekly about symptoms of acute respiratory tract infection (ARTI) during one RSV season. ARTI patients were tested for RSV during home visits and completed a daily symptom diary. RSV illness included PCR-confirmed ARTI and those showing seroconversion over the season. RSV ARTI was based on PCR alone (ClinicalTrials.gov, NCT03621930). RESULTS: We recruited 1040 participants (527 in season 2017-2018 and 513 in season 2018-2019) with a median age of 75 years (range 60-100 years). Of these, 1023 (99%) lived independently at home at baseline. RSV illness incidence was 22 out of 527 (4.2%) and 37 out of 513 (7.2%) in the respective seasons. RSV illness did not affect frailty or cardiopulmonary status during the course of the study. No patients were hospitalised or died from RSV illness. In the 36 patients with PCR confirmed RSV ARTI, symptom duration averaged 19 days, while a doctor's visit took place in 11 out of 36 cases (31%). RSV ARTI could not be differentiated clinically from all other ARTIs based on symptoms. CONCLUSION: This European study showed that RSV is prevalent in community-dwelling older adults and rarely causes severe disease. This suggests that watchful waiting, using a continuity of care approach to identify those who do need more intensive care, is often justified when RSV is suspected in family practice.

Biomarkers for Disease Severity in Children Infected With Respiratory Syncytial Virus: A Systematic Literature Review.

BACKGROUND: Clinical manifestations of respiratory syncytial virus (RSV) infection vary widely from mild, self-limiting illness to severe life-threatening disease. There are gaps in knowledge of biomarkers to objectively define severe disease and predict clinical outcomes. METHODS: A systematic search was performed, 1945-March 2019 in databases Ovid Medline, Embase, Global health, Scopus, and Web of Science. Risk of bias was assessed using the Cochrane tool. RESULTS: A total of 25 132 abstracts were screened and studies were assessed for quality, risk of bias, and extracted data; 111 studies met the inclusion criteria. RSV severity was correlated with antibody titers, reduced T and B cells, dysregulated innate immunity, neutrophil mobilization to the lungs and blood, decreased Th1 response, and Th2 weighted shift. Microbial exposures in respiratory tract may contribute to neutrophil mobilization to the lungs of the infants with severe RSV compared with mild RSV disease. CONCLUSIONS: Although a wide range of biomarkers have been associated with RSV disease severity, robust validated biomarkers are lacking. This review illustrates the broad heterogeneity of study designs and high variability in the definition of severe RSV disease. Prospective studies are required to validate biomarkers. Additional research investigating epigenetics, metabolomics, and microbiome holds promise for novel biomarkers.

Presumed Risk Factors and Biomarkers for Severe Respiratory Syncytial Virus Disease and Related Sequelae: Protocol for an Observational Multicenter, Case-Control Study From the Respiratory Syncytial Virus Consortium in Europe (RESCEU).

Respiratory syncytial virus (RSV) is the leading viral pathogen associated with acute lower respiratory tract infection and hospitalization in children < 5 years of age worldwide. While there are known clinical risk factors for severe RSV infection, the majority of those hospitalized are previously healthy infants. There is consequently an unmet need to identify biomarkers that predict host response, disease severity, and sequelae. The primary objective is to identify biomarkers of severe RSV acute respiratory tract infection (ARTI) in infants. Secondary objectives include establishing biomarkers associated with respiratory sequelae following RSV infection and characterizing the viral load, RSV whole-genome sequencing, host immune response, and transcriptomic, proteomic, metabolomic and epigenetic signatures associated with RSV disease severity. Six hundred thirty infants will be recruited across 3 European countries: the Netherlands, Spain, and the United Kingdom. Participants will be recruited into 2 groups: (1) infants with confirmed RSV ARTI (includes upper and lower respiratory tract infections), 500 without and 50 with comorbidities; and (2) 80 healthy controls. At baseline, participants will have nasopharyngeal, blood, buccal, stool, and urine samples collected, plus complete a questionnaire and 14-day symptom diary. At convalescence (7 weeks ± 1 week post-ARTI), specimen collection will be repeated. Laboratory measures will be correlated with symptom severity scores to identify corresponding biomarkers of disease severity. CLINICAL TRIALS REGISTRATION: NCT03756766.

Carbon Nanotube- and Asbestos-Induced DNA and RNA Methylation Changes in Bronchial Epithelial Cells.

Carbon nanotubes (CNTs) are nanoscale tube-shaped carbon materials used in many industrial areas. Their fiber shape has caused concerns about their toxicity given their structural similarity with asbestos. The aim here was to elucidate the effect of CNTs and asbestos exposure on global DNA and RNA methylation and the methylation of genes associated with cell cycle, inflammation, and DNA damage processes in human lung cells. Human bronchial epithelial cells (16HBE14o-) were exposed for 24 h to 25 and 100 µg/mL CNTs (single-walled CNTs [SWCNTs] and multiwalled CNTs [MWCNTs]) and 2.5 µg/mL asbestos (chrysotile, amosite, and crocidolite). Global DNA and RNA (hydroxy)methylation to cytosines was measured by a validated liquid chromatography tandem-mass spectrometry method. Global RNA methylation to adenines was measured by a colorimetric ELISA-like assay. Gene-specific DNA methylation status at certain cytosine-phosphate-guanine (CpG) sites of cyclin-dependent kinase inhibitor 1A ( CDKN1A), serine/threonine kinase ( ATM), and TNF receptor-associated factor 2 ( TRAF2) were analyzed by using bisulfite pyrosequencing technology. Only MWCNT-exposed cells showed significant global DNA hypomethylation of cytosine and global RNA hypomethylation of adenosine. SWCNT, MWCNT, and amosite exposure decreased DNA methylation of CDKN1A. ATM methylation was affected by chrysotile, SWCNT, and MWCNT. However, SWCNT exposure led to DNA hypermethylation of TRAF2. These findings contribute to further understanding of the effect of CNTs on different carcinogenic pathways.

Differences in MWCNT- and SWCNT-induced DNA methylation alterations in association with the nuclear deposition.

BACKGROUND: Subtle DNA methylation alterations mediated by carbon nanotubes (CNTs) exposure might contribute to pathogenesis and disease susceptibility. It is known that both multi-walled carbon nanotubes (MWCNTs) and single-walled carbon nanotubes (SWCNTs) interact with nucleus. Such, nuclear-CNT interaction may affect the DNA methylation effects. In order to understand the epigenetic toxicity, in particular DNA methylation alterations, of SWCNTs and short MWCNTs, we performed global/genome-wide, gene-specific DNA methylation and RNA-expression analyses after exposing human bronchial epithelial cells (16HBE14o- cell line). In addition, the presence of CNTs on/in the cell nucleus was evaluated in a label-free way using femtosecond pulsed laser microscopy. RESULTS: Generally, a higher number of SWCNTs, compared to MWCNTs, was deposited at both the cellular and nuclear level after exposure. Nonetheless, both CNT types were in physical contact with the nuclei. While particle type dependency was noticed for the identified genome-wide and gene-specific alterations, no global DNA methylation alteration on 5-methylcytosine (5-mC) sites was observed for both CNTs. After exposure to MWCNTs, 2398 genes were hypomethylated (at gene promoters), and after exposure to SWCNTs, 589 CpG sites (located on 501 genes) were either hypo- (N = 493 CpG sites) or hypermethylated (N = 96 CpG sites). Cells exposed to MWCNTs exhibited a better correlation between gene promoter methylation and gene expression alterations. Differentially methylated and expressed genes induced changes (MWCNTs > SWCNTs) at different cellular pathways, such as p53 signalling, DNA damage repair and cell cycle. On the other hand, SWCNT exposure showed hypermethylation on functionally important genes, such as SKI proto-oncogene (SKI), glutathione S-transferase pi 1 (GTSP1) and shroom family member 2 (SHROOM2) and neurofibromatosis type I (NF1), which the latter is both hypermethylated and downregulated. CONCLUSION: After exposure to both types of CNTs, epigenetic alterations may contribute to toxic or repair response. Moreover, our results suggest that the observed differences in the epigenetic response depend on particle type and differential CNT-nucleus interactions.

Epigenetic effects of carbon nanotubes in human monocytic cells.

Carbon nanotubes (CNTs) are fibrous carbon-based nanomaterials with a potential to cause carcinogenesis in humans. Alterations in DNA methylation on cytosine-phosphate-guanidine (CpG) sites are potential markers of exposure-induced carcinogenesis. This study examined cytotoxicity, genotoxicity and DNA methylation alterations on human monocytic cells (THP-1) after incubation with single-walled CNTs (SWCNTs) and multi-walled CNTs (MWCNTs). Higher cytotoxicity and genotoxicity were observed after incubation with SWCNTs than incubation with MWCNTs. At the selected concentrations (25 and 100 µg/ml), DNA methylation alterations were studied. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to assess global DNA methylation, and Illumina 450K microarrays were used to assess methylation of single CpG sites. Next, we assessed gene promoter-specific methylation levels. We observed no global methylation or hydroxymethylation alterations, but on gene-specific level, distinct clustering of CNT-treated samples were noted. Collectively, CNTs induced gene promoter-specific altered methylation and those 1127 different genes were identified to be hypomethylated. Differentially methylated genes were involved in several signalling cascade pathways, vascular endothelial growth factor and platelet activation pathways. Moreover, possible contribution of the epigenetic alterations to monocyte differentiation and mixed M1/M2 macrophage polarisation were discussed.

Maternal serum alpha-fetoprotein levels are normal in Fanconi anemia: Can it be a lack of postnatal inhibition of AFP gene resulting in the elevation?

We investigated the feasibility of using serum alpha-fetoprotein (AFP) levels as a screening test for prenatal diagnosis of Fanconi anemia (FA). Serial measurements in maternal serum were recorded. Parents, both heterozygous for FA, had declined prenatal molecular testing. The infant was born with no somatic abnormalities, and FA was confirmed by postnatal molecular analysis. Maternal serum AFP levels during each trimester of pregnancy were normal indicating that these levels cannot be used as a screening test in prenatal diagnosis. Three-year follow-up after birth showed constantly elevated serum levels in the patient from the start, suggesting a lack of postnatal inhibition on AFP gene.

What have we learned from animal studies of immune responses to respiratory syncytial virus infection?

Respiratory syncytial virus (RSV) is a common cause of severe respiratory tract infection at the extremes of age and in vulnerable populations. However, it is difficult to predict the clinical course and most infants who develop severe disease have no pre-existing risk factors. With the recent licencing of RSV vaccines and monoclonal antibodies, it is important to identify high-risk individuals in order to prioritise those who will most benefit from prophylaxis. The immune response to RSV and the mechanisms by which the virus prevents the establishment of immunological memory have been extensively investigated but remain incompletely characterised. In animal models, beneficial and harmful immune responses have both been demonstrated. While only chimpanzees are fully permissive for human RSV replication, most research has been conducted in rodents, or in calves infected with bovine RSV. Based on these studies, components of innate and adaptive immune systems, cytokines, chemokines and metabolites, and specific genetic and transcriptomic signatures are identified as potential predictive indicators of RSV disease severity. These findings may inform the development of future human studies and contribute to the early identification of patients at high risk of severe infection. This narrative review summarises the factors involved in the immune response to RSV infection in these models and highlights the relationship between potential biomarkers and disease severity.

Genetic Profiling of Pediatric Patients with B-Cell Precursor Acute Lymphoblastic Leukemia.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous leukemia subgroup. It has multiple sub-types that are likely to be classified by prognostic factors. Following a systematic literature review, this study analyzed the genes correlated with BCP-ALL prognosis ( IKZF1, PAX5, EBF1, CREBBP, CRLF2, JAK2, ERG, CXCR4, ZAP70, VLA4, NF1, NR3C1, RB1, TSLP, ZNRF1, and FOXO3A) , specifically their nucleotide variations and expression profiles in pediatric BCP-ALL samples. The study included 45 pediatric BCP-ALL patients with no cytogenetic anomaly and a control group of 10 children. The selected genes' hot-spot regions were sequenced using next-generation sequencing, while Polymorphism Phenotyping v2 and Supplemental Nutrition Assistance Program were used to identify pathogenic mutations. The expression analysis was performed using quantitative real-time polymerase chain reaction. The mutation analysis detected 328 variants (28 insertions, 47 indels, 74 nucleotide variants, 75 duplications, and 104 deletions). The most and least frequently mutated genes were IKZF1 and CREBBP , respectively. There were statistically significant differences between patients and controls for mutation distribution in eight genes ( ERG, CRLF2, CREBBP, TSLP, JAK2, ZAP70, FOXO3A, and NR3C1 ). The expression analysis revealed that JAK and ERG were significantly overexpressed in patients compared with controls (respectively, p = 0.004 and p = 0.003). This study combined genes and pathways previously analyzed in pediatric BCP-ALL into one dataset for a comprehensive analysis from the same samples to unravel candidate prognostic biomarkers. Novel mutations were identified in all of the studied genes.

Has the Covid-19 Pandemic increased the complication rate in patients with acute appendicitis?

AIM: The aim of this study is to compare the numbers of patients, clinical outcomes, and complication rates of acute appendicitis before and after COVID-19 pandemic in our clinic. MATERIAL AND METHOD: This is a retrospective clinical study. Patients of 19 to 88 years of age that underwent emergency surgery with the diagnosis of acute appendicitis at Ankara City Hospital Department of General Surgery between 11 December 2019 and 11 June 2020 were included. The first case of COVID-19 in Turkey was announced on 11 March 2020. We studied the demographics, surgical procedures, and complication rates in 3 months periods before and after the first case was announced. RESULTS: A total number of 462 patients were analyzed between the ages of 19-88, 184 of which (39.8%) were females and 278 were males (60.2%). 253 of these patients were diagnosed with AA and underwent surgery before March 11 whereas 209 patients were diagnosed and treated after March 11. DISCUSSION: There was no statistical difference between the two groups in terms of complication rates before and after the pandemic. Although the rate of open appendectomy was increased after the pandemic, no statistical difference has been found. CONCLUSION: No change was observed in terms of hospital admissions, methods of treatment, complication rates, length of stay before and after the COVID-19 pandemic. KEY WORDS: Acute Appendicitis, Appendectomy, COVID-19.

T cells, more than antibodies, may prevent symptoms developing from respiratory syncytial virus infections in older adults.

Introduction: The immune mechanisms supporting partial protection from reinfection and disease by the respiratory syncytial virus (RSV) have not been fully characterized. In older adults, symptoms are typically mild but can be serious in patients with comorbidities when the infection extends to the lower respiratory tract. Methods: This study formed part of the RESCEU older-adults prospective-cohort study in Northern Europe (2017-2019; NCT03621930) in which a thousand participants were followed over an RSV season. Peripheral-blood samples (taken pre-season, post-season, during illness and convalescence) were analyzed from participants who (i) had a symptomatic acute respiratory tract infection by RSV (RSV-ARTI; N=35) or (ii) asymptomatic RSV infection (RSV-Asymptomatic; N=16). These analyses included evaluations of antibody (Fc-mediated-) functional features and cell-mediated immunity, in which univariate and machine-learning (ML) models were used to explore differences between groups. Results: Pre-RSV-season peripheral-blood biomarkers were predictive of symptomatic RSV infection. T-cell data were more predictive than functional antibody data (area under receiver operating characteristic curve [AUROC] for the models were 99% and 76%, respectively). The pre-RSV season T-cell phenotypes which were selected by the ML modelling and which were more frequent in RSV-Asymptomatic group than in the RSV-ARTI group, coincided with prominent phenotypes identified during convalescence from RSV-ARTI (e.g., IFN-γ+, TNF-α+ and CD40L+ for CD4+, and IFN-γ+ and 4-1BB+ for CD8+). Conclusion: The evaluation and statistical modelling of numerous immunological parameters over the RSV season suggests a primary role of cellular immunity in preventing symptomatic RSV infections in older adults.

Single-cell immune profiling reveals markers of emergency myelopoiesis that distinguish severe from mild respiratory syncytial virus disease in infants.

Whereas most infants infected with respiratory syncytial virus (RSV) show no or only mild symptoms, an estimated 3 million children under five are hospitalized annually due to RSV disease. This study aimed to investigate biological mechanisms and associated biomarkers underlying RSV disease heterogeneity in young infants, enabling the potential to objectively categorize RSV-infected infants according to their medical needs. Immunophenotypic and functional profiling demonstrated the emergence of immature and progenitor-like neutrophils, proliferative monocytes (HLA-DRLow , Ki67+), impaired antigen-presenting function, downregulation of T cell response and low abundance of HLA-DRLow B cells in severe RSV disease. HLA-DRLow monocytes were found as a hallmark of RSV-infected infants requiring hospitalization. Complementary transcriptomics identified genes associated with disease severity and pointed to the emergency myelopoiesis response. These results shed new light on mechanisms underlying the pathogenesis and development of severe RSV disease and identified potential new candidate biomarkers for patient stratification.

Distinct patterns of within-host virus populations between two subgroups of human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in young children globally, but little is known about within-host RSV diversity. Here, we characterised within-host RSV populations using deep-sequencing data from 319 nasopharyngeal swabs collected during 2017-2020. RSV-B had lower consensus diversity than RSV-A at the population level, while exhibiting greater within-host diversity. Two RSV-B consensus sequences had an amino acid alteration (K68N) in the fusion (F) protein, which has been associated with reduced susceptibility to nirsevimab (MEDI8897), a novel RSV monoclonal antibody under development. In addition, several minor variants were identified in the antigenic sites of the F protein, one of which may confer resistance to palivizumab, the only licensed RSV monoclonal antibody. The differences in within-host virus populations emphasise the importance of monitoring for vaccine efficacy and may help to explain the different prevalences of monoclonal antibody-escape mutants between the two subgroups.

Induction and recovery of CpG site specific methylation changes in human bronchial cells after long-term exposure to carbon nanotubes and asbestos.

INTRODUCTION: Inhalation of asbestos induces lung cancer via different cellular mechanisms. Together with the increased production of carbon nanotubes (CNTs) grows the concern about adverse effects on the lungs given the similarities with asbestos. While it has been established that CNT and asbestos induce epigenetic alterations, it is currently not known whether alterations at epigenetic level remain stable after withdrawal of the exposure. Identification of DNA methylation changes after a low dose of CNT and asbestos exposure and recovery can be useful to determine the fibre/particle toxicity and adverse outcome. METHODS: Human bronchial epithelial cells (16HBE) were treated with a low and non-cytotoxic dose (0.25 µg/ml) of multi-walled carbon nanotubes (MWCNTs-NM400) or single-walled carbon nanotubes (SWCNTs-SRM2483) and 0.05 µg/ml amosite (brown) asbestos for the course of four weeks (sub-chronic exposure). After this treatment, the cells were further incubated (without particle/fibre) for two weeks, allowing recovery from the exposure (recovery period). Nuclear depositions of the CNTs were assessed using femtosecond pulsed laser microscopy in a label-free manner. DNA methylation alterations were analysed using microarrays that assess more than 850 thousand CpG sites in the whole genome. RESULTS: At non-cytotoxic doses, CNTs were noted to be incorporated with in the nucleus after a four weeks period. Exposure to MWCNTs induced a single hypomethylation at a CpG site and gene promoter region. No change in DNA methylation was observed after the recovery period for MWCNTs. Exposure to SWCNTs or amosite induced hypermethylation at CpG sites after sub-chronic exposure which may involve in 'transcription factor activity' and 'sequence-specific DNA binding' gene ontologies. After the recovery period, hypermethylation and hypomethylation were noted for both SWCNTs and amosite. Hippocalcinlike 1 (HPCAL1), protease serine 3 (PRSS3), kallikrein-related peptidase 3 (KLK3), kruppel like factor 3 (KLF3) genes were hypermethylated at different time points in either SWCNT-exposed or amosite-exposed cells. CONCLUSION: These results suggest that the specific SWCNT (SRM2483) and amosite fibres studied induce hypo- or hypermethylation on CpG sites in DNA after very low-dose exposure and recovery period. This effect was not seen for the studied MWCNT (NM400).