nf-core/airrflow: an adaptive immune receptor repertoire analysis workflow employing the Immcantation framework (preprint)

MotivationAdaptive Immune Receptor Repertoire sequencing (AIRR-seq) is a valuable experimental tool to study the immune state in health and following immune challenges such as infectious diseases, (auto)immune diseases, and cancer. Several tools have been developed to reconstruct B cell and T cell receptor sequences from AIRR-seq data and infer B and T cell clonal relationships. However, currently available tools offer limited parallelization across samples, scalability or portability to high-performance computing infrastructures. ResultsTo address this need, we developed nf-core/airrflow, an end-to-end bulk and single-cell AIRR-seq processing workflow which integrates the Immcantation Framework following BCR and TCR sequencing data analysis best practices. The Immcantation Framework is a comprehensive toolset, which allows the processing of bulk and single-cell AIRR-seq data from raw read processing to clonal inference. nf-core/airrflow is written in Nextflow and is part of the nf-core project, which collects community contributed and curated Nextflow workflows for a wide variety of analysis tasks. We assessed the performance of nf-core/airrflow on simulated sequencing data and show example results with real datasets. To demonstrate the applicability of nf-core/airrflow to the high-throughput processing of large AIRR-seq datasets, we validated and extended previously reported findings of convergent antibody responses to SARS-CoV-2 by analyzing 97 COVID-19 infected individuals and 99 healthy controls retrieved from public databases. Availability and implementationnf-core/airrflow is available free of charge, under the MIT license on GitHub (https://github.com/nf-core/airrflow). Documentation and example results are available at https://nf-co.re/airrflow. Visual abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/576147v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@8a3352org.highwire.dtl.DTLVardef@12f77daorg.highwire.dtl.DTLVardef@165a897org.highwire.dtl.DTLVardef@11f8ebe_HPS_FORMAT_FIGEXP M_FIG C_FIG

Distinct profiles and kinetics of soluble infammatory markers in patients with acute and chronic HCV-infections and SARS-CoV-2-infections: Implications for the long-term consequences of viral infections?

Background and Aims Viral infections occur acutely but can also progress chronically, with the immune system having a central role in immunopathoge-nesis. The question arises whether all alterations in immune responses are reversible after viral elimination (spontaneously or by therapy). Therefore, the aim of this study is to compare soluble infammatory markers (SIM) during and after infection with SARS-CoV-2 and acute and chronic HCV-infections. Patients and Method Patients with acute HCV (n = 29), chronic HCV (n = 54), SARS-CoV-2 (n = 39) and 31 healthy-controls were included. Blood samples were tested at baseline, end of treatment/infection, and follow-up ( >= 9 months after baseline). IL-12p70, IL-1b, IL-4, IL-5, IL-6, IL-8, TNF, IFN-g, IL-10, IL-22, CXCL-10, MCP-1, MIP-1b, ITAC were quantified using the HD-SP-X Imaging and Analysis SystemTM. Results SIM profiles in patients with acute HCV were substantially elevated at baseline and the decrease during follow-up was considerably less compared to the SARS-CoV-2 cohort. In chronic HCV-patients, viral elimination by therapy resulted in a decrease in SIM, although not always to those of controls. Cirrhotic HCV patients had higher SIM levels after HCV elimination than non-cirrhotic chronic HCV-patients. In the SARS-CoV-2 cohort, most SIM returned to levels of controls 3 months after baseline. Conclusions SIM profiles and kinetics after viral elimination difer between blood-borne acute and chronic HCV- and respiratory SARS-CoV-2-infections. The immunologic imprint 9 months after cured HCV-infection (both acute and chronic) appears to be more pronounced than after SARS-CoV-2-infection. Further analysis is needed to correlate the SIM profle with the clinical pheno-type (long-HepC vs. long-COVID-19).

Negative association between fatigue and signs of sleep apnoea in patients after COVID-19.

Compared to the general population, there are increased apnoea-hypopnoea indices in patients recovering from #COVID19, yet there is a negative correlation to symptoms of fatigue and no significant correlation to daytime sleepiness https://bit.ly/3pEl9C8.

In Vitro Screening Identifies TRPV4 and PAR1 as Targets for Endothelial Barrier Stabilization in COVID‐19

Study objective Endothelial dysfunction and increased microvascular permeability are hallmarks of severe COVID‐19. At present, the underlying mechanisms of endothelial barrier failure in COVID‑19 remain elusive. Here, we show that increased thrombin activity in plasma from severe COVID‐19 patients activates endothelial protease‐activated receptor (PAR1), which mediates barrier failure by triggering TRPV4‐mediated Ca2+ influx in lung microvascular endothelial cells. Methods Citrate plasma was sampled as part of the Pa‐COVID‐19 cohort study (ethics approval EA2/066/20) from patients with severe COVID‐19 (high flow O2 or mechanically ventilated;WHO severity score: 5‐7) COVID‑19. Plasma samples were diluted to 10% (v/v) in cell culture medium without FCS and tested for their ability to disrupt barrier integrity of primary human pulmonary microvascular endothelial cells (HPMEC) monolayers by electrical cell‐substrate impedance sensing (ECIS), immunofluorescence for endothelial VE‐cadherin and F‐actin, western blot analyses of PAR‐1 cleavage, and real‐time Ca2+ imaging. Plasma from healthy donors served as control. Results COVID‐19 plasma had elevated thrombin activity while levels of antithrombin III, a key anti‐coagulant with thromboprotective function were decreased. COVID‐19 plasma caused endothelial barrier dysfunction as measured by ECIS and gap formation in HPMEC monolayers. Endothelial barrier disruption and endothelial Ca2+ influx in response to COVID‐19 plasma could be blocked by selective antagonists targeting thrombin (Argatroban), its receptor PAR1 (SCH79797), or TRPV4 (HC‐067047). Conclusion Here, we identify a novel signaling axis involving thrombin, its receptor PAR1, and TRPV4 as mechanism for increased microvascular permeability in COVID‑19. Targeting this signaling axis in endothelial barrier failure may provide a promising adjunctive therapy in COVID‐19.

Altered fibrin clot structure and dysregulated fibrinolysis contribute to thrombosis risk in severe COVID-19.

The high incidence of thrombotic events suggests a possible role of the contact system pathway in COVID-19 pathology. In this study, we determined the altered levels of factor XII (FXII) and its activation products in critically ill patients with COVID-19 in comparison with patients with severe acute respiratory distress syndrome related to the influenza virus (acute respiratory distress syndrome [ARDS]-influenza). Compatible with those data, we found rapid consumption of FXII in COVID-19 but not in ARDS-influenza plasma. Interestingly, the lag phase in fibrin formation, triggered by the FXII activator kaolin, was not prolonged in COVID-19, as opposed to that in ARDS-influenza. Confocal and electron microscopy showed that increased FXII activation rate, in conjunction with elevated fibrinogen levels, triggered formation of fibrinolysis-resistant, compact clots with thin fibers and small pores in COVID-19. Accordingly, clot lysis was markedly impaired in COVID-19 as opposed to that in ARDS-influenza. Dysregulated fibrinolytic system, as evidenced by elevated levels of thrombin-activatable fibrinolysis inhibitor, tissue-plasminogen activator, and plasminogen activator inhibitor-1 in COVID-19 potentiated this effect. Analysis of lung tissue sections revealed widespread extra- and intravascular compact fibrin deposits in patients with COVID-19. A compact fibrin network structure and dysregulated fibrinolysis may collectively contribute to a high incidence of thrombotic events in COVID-19.

Altered fibrin clot structure contributes to thrombosis risk in severe COVID-19 (preprint)

The high incidence of thrombotic events suggests a possible role of the contact system pathway in COVID-19 pathology. Here, we demonstrate altered levels of factor XII (FXII) and its activation products in two independent cohorts of critically ill COVID-19 patients in comparison to patients suffering from severe acute respiratory distress syndrome due to influenza virus (ARDS-influenza). Compatible with this data, we report rapid consumption of FXII in COVID-19, but not in ARDS-influenza, plasma. Interestingly, the kaolin clotting time was not prolonged in COVID-19 as compared to ARDS-influenza. Using confocal and electron microscopy, we show that increased FXII activation rate, in conjunction with elevated fibrinogen levels, triggers formation of fibrinolysis-resistant, compact clots with thin fibers and small pores in COVID-19. Accordingly, we observed clot lysis in 30% of COVID-19 patients and 84% of ARDS-influenza subjects. Analysis of lung tissue sections revealed wide-spread extra- and intra-vascular compact fibrin deposits in COVID-19. Together, our results indicate that elevated fibrinogen levels and increased FXII activation rate promote thrombosis and thrombolysis resistance via enhanced thrombus formation and stability in COVID-19.

Effectiveness of a Three-Week Inpatient Pulmonary Rehabilitation Program for Patients after COVID-19: A Prospective Observational Study.

For COVID-19 patients who remain symptomatic after the acute phase, pulmonary rehabilitation (PR) is recommended. However, only a few studies have investigated the effectiveness of PR, especially considering the duration between the acute phase of COVID-19 and the onset of rehabilitation, as well as the initial severity. This prospective observational study evaluated the efficacy of PR in patients after COVID-19. A total of 120 still-symptomatic patients referred for PR after overcoming acute COVID-19 were asked to participate, of whom 108 (mean age 55.6 ± 10.1 years, 45.4% female) consented. The patients were assigned to three groups according to the time of referral and initial disease severity (severe acute; severe after interval; mild after interval). The primary outcome was dyspnea. Secondary outcomes included other respiratory disease symptoms, physical capacity, lung function, fatigue, quality of life (QoL), depression, and anxiety. Furthermore, patients rated the overall effectiveness of PR and their subjective change in health status. At the end of PR, we detected improvements with large effect sizes in exertional dyspnea, physical capacity, QoL, fatigue, and depression in the overall group. Other parameters changed with small to medium effect sizes. PR was effective after acute COVID-19 in all three groups analyzed.

Impact of the COVID-19 pandemic on inflammatory bowel disease: The role of emotional stress and social isolation.

Inflammatory bowel disease (IBD) is a chronic health condition exacerbated by negative emotional stress experiences. In the current study, we examined whether the outbreak of the COVID-19 pandemic coincided with an increase in stress experiences and accordingly an aggravation of disease activity in IBD patients. Sixty-three IBD patients (30 Crohn's disease or CD, 33 ulcerative colitis) completed an online survey during the COVID-19-related lockdown, assessing clinical disease activity, disease-related quality of life, presence of functional gastrointestinal symptoms, social isolation and stress experiences. Scores were then compared to pre-lockdown baseline screening. The pandemic yielded a significant baseline-to-lockdown increase in emotional stress and social isolation. Stress increments, particularly those occasioned by interpersonal tension and excessive interpersonal proximity, were associated with a worsening of functional gastrointestinal symptoms. Exacerbations of loneliness coincided with an escalation of CD activity, functional gastrointestinal symptoms and a decline in subjective health. Lastly, COVID-19 anxiety was significantly related to CD symptom severity and social dysfunction. The findings show that shifts in IBD expression are closely linked to changes in emotional stress experiences and interpersonal relatedness. As such, they contribute to a better understanding of inter-individual differences in IBD progression and provide leads for therapeutic interventions.

Short-term Effects of a Three-week Inpatient Post-COVID-19 Pulmonary Rehabilitation Program - a Prospective Observational Study (preprint)

Background:The clinical course of coronavirus disease 2019 (COVID-19) varies distinctly. Particularly after severe or critical courses, i.e., after hospitalization, clinical manifestations frequently persist after the acute phase. However, symptoms may also persist after initially milder courses that can be treated in an outpatient setting. For patients who remain symptomatic after COVID-19, pulmonary rehabilitation (PR) is recommended. However, only few studies investigated the effectiveness of PR, especially considering different disease courses. The main objective was to evaluate the feasibility, safety, and efficacy of post-COVID-19 PR.Methods:A total of 120 post-COVID-19 patients who were referred to the Bad Reichenhall Clinic between April 2020 and January 2021 were asked to participate in this prospective observational study. PR was tailored to each patient’s individual needs and was based on the current recommendations. The primary outcome dyspnea was assessed with numerical rating scales and the modified Medical Research Council (mMRC) dyspnea scale. Secondary outcomes included other symptoms such as cough and sputum; physical capacity; lung function; fatigue; quality of life (QoL); depression; and anxiety. Furthermore, patients rated the overall effectiveness of PR and their subjective change in health status.Results:A total of 108 patients (mean age 55.6±10.1 years, 45.4% female) were included and were assigned to 3 groups depending on the referral mode (A: severe acute; B: severe after interval, C: mild after interval). At the end of PR, we detected improvements in the intensity of exertional dyspnea, physical capacity, QoL, fatigue, and depression in the overall group, with large effect sizes (Cohen’s d>0.8). Moderate effect sizes (0.5≤d<0.8) were found for resting dyspnea and the mMRC-dyspnea scale. Cohen’s d>0.4 was found for vital capacity, forced expiratory volume in one second, partial oxygen pressure, and anxiety. Significant but rather small effect sizes (0.2<d<0.4) were found for cough, sputum, pain, and other lung function parameters.Conclusions:PR is feasible, safe, and effective after acute COVID-19, which was true for all 3 groups analyzed, with a trend toward greater efficacy after severe courses of COVID-19 and an earlier start of PR after acute COVID-19. Therefore, all post-COVID-19 patients who remain symptomatic should be offered PR.Trial registration: German Clinical Trials Register, DRKS00023180. Registered 01 September 2020 – Retrospectively registered, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00023180