Immunostimulatory Effect of Flagellin on MDR-Klebsiella-Infected Human Airway Epithelial Cells.

Pneumonia caused by multi-drug-resistant Klebsiella pneumoniae (MDR-Kpneu) poses a major public health threat, especially to immunocompromised or hospitalized patients. This study aimed to determine the immunostimulatory effect of the Toll-like receptor 5 ligand flagellin on primary human lung epithelial cells during infection with MDR-Kpneu. Human bronchial epithelial (HBE) cells, grown on an air-liquid interface, were inoculated with MDR-Kpneu on the apical side and treated during ongoing infection with antibiotics (meropenem) and/or flagellin on the basolateral and apical side, respectively; the antimicrobial and inflammatory effects of flagellin were determined in the presence or absence of meropenem. In the absence of meropenem, flagellin treatment of MDR-Kpneu-infected HBE cells increased the expression of antibacterial defense genes and the secretion of chemokines; moreover, supernatants of flagellin-exposed HBE cells activated blood neutrophils and monocytes. However, in the presence of meropenem, flagellin did not augment these responses compared to meropenem alone. Flagellin did not impact the outgrowth of MDR-Kpneu. Flagellin enhances antimicrobial gene expression and chemokine release by the MDR-Kpneu-infected primary human bronchial epithelium, which is associated with the release of mediators that activate neutrophils and monocytes. Topical flagellin therapy may have potential to boost immune responses in the lung during pneumonia.

Long-term evolution of human seasonal influenza virus A(H3N2) is associated with an increase in polymerase complex activity.

Since the influenza pandemic in 1968, influenza A(H3N2) viruses have become endemic. In this state, H3N2 viruses continuously evolve to overcome immune pressure as a result of prior infection or vaccination, as is evident from the accumulation of mutations in the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, phylogenetic studies have also demonstrated ongoing evolution in the influenza A(H3N2) virus RNA polymerase complex genes. The RNA polymerase complex of seasonal influenza A(H3N2) viruses produces mRNA for viral protein synthesis and replicates the negative sense viral RNA genome (vRNA) through a positive sense complementary RNA intermediate (cRNA). Presently, the consequences and selection pressures driving the evolution of the polymerase complex remain largely unknown. Here, we characterize the RNA polymerase complex of seasonal influenza A(H3N2) viruses representative of nearly 50 years of influenza A(H3N2) virus evolution. The H3N2 polymerase complex is a reassortment of human and avian influenza virus genes. We show that since 1968, influenza A(H3N2) viruses have increased the transcriptional activity of the polymerase complex while retaining a close balance between mRNA, vRNA, and cRNA levels. Interestingly, the increased polymerase complex activity did not result in increased replicative ability on differentiated human airway epithelial (HAE) cells. We hypothesize that the evolutionary increase in polymerase complex activity of influenza A(H3N2) viruses may compensate for the reduced HA receptor binding and avidity that is the result of the antigenic evolution of influenza A(H3N2) viruses.

Inflammatory profiles are associated with long COVID up to 6 months after COVID-19 onset: A prospective cohort study of individuals with mild to critical COVID-19.

BACKGROUND: After initial COVID-19, immune dysregulation may persist and drive post-acute sequelae of COVID-19 (PASC). We described longitudinal trajectories of cytokines in adults up to 6 months following SARS-CoV-2 infection and explored early predictors of PASC. METHODS: RECoVERED is a prospective cohort of individuals with laboratory-confirmed SARS-CoV-2 infection between May 2020 and June 2021 in Amsterdam, the Netherlands. Serum was collected at weeks 4, 12 and 24 of follow-up. Monthly symptom questionnaires were completed from month 2 after COVID-19 onset onwards; lung diffusion capacity (DLCO) was tested at 6 months. Cytokine concentrations were analysed by human magnetic Luminex screening assay. We used a linear mixed-effects model to study log-concentrations of cytokines over time, assessing their association with socio-demographic and clinical characteristics that were included in the model as fixed effects. RESULTS: 186/349 (53%) participants had ≥2 serum samples and were included in current analyses. Of these, 101/186 (54%: 45/101[45%] female, median age 55 years [IQR = 45-64]) reported PASC at 12 and 24 weeks after COVID-19 onset. We included 37 reference samples (17/37[46%] female, median age 49 years [IQR = 40-56]). In a multivariate model, PASC was associated with raised CRP and abnormal diffusion capacity with raised IL10, IL17, IL6, IP10 and TNFα at 24 weeks. Early (0-4 week) IL-1ß and BMI at COVID-19 onset were predictive of PASC at 24 weeks. CONCLUSIONS: Our findings indicate that immune dysregulation plays an important role in PASC pathogenesis, especially among individuals with reduced pulmonary function. Early IL-1ß shows promise as a predictor of PASC.

Neuropsychological functioning after COVID-19: minor differences between individuals with and without persistent complaints after SARS-CoV-2 infection.

Objective: It is unclear how self-reported severe fatigue and difficulty concentrating after SARS-CoV-2 infection relate to objective neuropsychological functioning. The study aimed to compare neuropsychological functioning between individuals with and without these persistent subjective complaints. Method: Individuals with and without persistent severe fatigue (Checklist Individual Strength (CIS) fatigue ≥ 35) and difficulty concentrating (CIS concentration ≥ 18) at least 3 months after SARS-CoV-2 infection were included. Neuropsychological assessment was performed on overall cognitive functioning, attention, processing speed, executive functioning, memory, visuo-construction, and language (18 tests). T-scores -1.5 SD below population normative data (T ≤ 35) were classified as "impaired". Results: 230 participants were included in the study, of whom 22 were excluded from the analysis due to invalid performance. Of the participants included in the analysis, 111 reported persistent complaints of severe fatigue and difficulty concentrating and 97 did not. Median age was 54 years, 59% (n = 126) were female, and participants were assessed a median of 23 months after first infection (IQR: 16-28). With bivariate logistic regression, individuals with persistent complaints had an increased likelihood of slower information processing speed performance on the Stroop word reading (OR = 2.45, 95%CI = 1.02-5.84) compared to those without persistent complaints. Demographic or clinical covariates (e.g. hospitalization) did not influence this association. With linear regression techniques, persistent complaints were associated with lower t-scores on the D2 CP, TMT B, and TMT B|A. There were no differences in performance on the other neuropsychological tests. Conclusions: Individuals with subjective severe fatigue and difficulty concentrating after COVID-19 do not typically demonstrate cognitive impairment on extensive neuropsychological testing.

Determinants of epidemic size and the impacts of lulls in seasonal influenza virus circulation.

During the COVID-19 pandemic, levels of seasonal influenza virus circulation were unprecedentedly low, leading to concerns that a lack of exposure to influenza viruses, combined with waning antibody titres, could result in larger and/or more severe post-pandemic seasonal influenza epidemics. However, in most countries the first post-pandemic influenza season was not unusually large and/or severe. Here, based on an analysis of historical influenza virus epidemic patterns from 2002 to 2019, we show that historic lulls in influenza virus circulation had relatively minor impacts on subsequent epidemic size and that epidemic size was more substantially impacted by season-specific effects unrelated to the magnitude of circulation in prior seasons. From measurements of antibody levels from serum samples collected each year from 2017 to 2021, we show that the rate of waning of antibody titres against influenza virus during the pandemic was smaller than assumed in predictive models. Taken together, these results partially explain why the re-emergence of seasonal influenza virus epidemics was less dramatic than anticipated and suggest that influenza virus epidemic dynamics are not currently amenable to multi-season prediction.