Decreased DNase activity in severe COVID-19 correlates with increased circulating NET burden C. Martens1

Background: COVID-19 represents a perfect storm for release of neutrophil extracellular traps (NETs) and resultant pathology from immunothrombosis. Levels of NETs in circulation are regulated by endogenous plasma DNases, which have been shown to be reduced in various diseases including myocardial infarction and sepsis. We previously reported elevated NET biomarkers in admission samples from our first wave study cohort. Aim(s): To characterize DNase activity and biomarkers of released NETs in the context of COVID-19 immunothrombosis. Method(s): With ethical permission and informed consent, we prospectively collected citrated platelet-poor plasma samples from patients admitted to the COVID ward (55 patients) or intensive care unit (216 patients) from March 2020-December 2021 as part of the COntAGIouS trial at UZ Leuven in Belgium (NCT04327750), with special attention paid to sample preparation and storage to preserve NET fragments and DNase activity. Consecutive samples were obtained within 48 hours of admission, between days 6-8, and upon hospital or ICU discharge. Analysis was batch-performed for MPO, MPO-DNA, PF4, sP-selectin, citrullinated histones, DNase activity, VWF:Ag, and FVIII:Ag levels. Result(s): In ICU patients, MPO, VWF, sP-selectin, and NET biomarkers were elevated throughout hospitalization, peaking at day 6-8 after admission, whereas PF4 and FVIII remained highly elevated through the time of ICU discharge. DNase activity was decreased in admission samples, normalized at day 6-8, and strongly increased at the time of discharge, indicating a potential compensatory mechanism. DNase activity was negatively correlated with MPO-DNA values (r = -0.29, p = 0.0013). sPselectin and NET levels were significantly higher in admission samples for patients who experienced a thrombotic event in the period during hospitalization, including pulmonary embolism, DVT, myocardial infarction, and/or stroke. Conclusion(s): Elevated NET levels and decreased DNase activity in plasma are correlated in severe COVID-19, together with elevated markers of thrombotic risk. Approaches to restore DNase activity in plasma may be beneficial in COVID-19-associated immunothrombosis.

The kallikrein-kinin system in bronchoalveolar lavage fluid from patients with COVID-19

Background : Markers of both inflammation and coagulation are linked to clinical outcome in coronavirus disease 2019 (COVID-19). Binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the angiotensin-converting enzyme 2 receptor, which is involved in kinin breakdown, interferes with the kallikreinkinin pathway. This could result in increased vascular permeability, fluid excess in the lungs and pulmonary edema. Furthermore, the kallikrein-kinin pathway links coagulation and inflammation through its interactions with the contact activation pathway of coagulation via factor XII and with neutrophil extracellular traps (NETs). These insights could help to explain the clinical presentation of COVID-19 pneumonia with pulmonary coagulopathy and the high incidence of thromboembolic complications in COVID-19. Aims : Given the lack of clinical evidence to support this hypothesis, we studied the kallikrein-kinin system in bronchoalveolar lavage (BAL) fluid. Methods : In BAL fluid samples from patients with or without COVID-19, we performed in-depth analyses of kinin peptides (bradykinin, Lys-bradykinin, Lys-bradykinin-(1-8), bradykinin-(1-8), bradykinin-(1-7), and bradykinin-(1-5)) using a liquid chromatography with tandem mass spectrometry assay, along with measurements of plasma and tissue kallikrein hydrolytic activity and myeloperoxidase (MPO)-DNA complexes as a biomarker for NETs. Informed consent and ethical approval were obtained. Results : We observed higher levels of the most downstream kinin peptide bradykinin-(1-5) (Figure 1), higher tissue kallikrein activity (Figure 2), and higher levels of MPO-DNA complexes (699.0 ng/mL [66.0-142621.0], median [range], n = 21 vs 70.5 [9.9-960.0], n = 19;P < 0.001) in BAL fluid from patients with COVID-19 compared to those in BAL fluid from patients without COVID-19. Conclusions : Our data support the hypothesis that SARS-CoV-2 induces dysregulation of the kallikrein-kinin system, which contributes to thromboinflammation in COVID-19. These findings encourage the investigation of drugs that target the kallikrein-kinin system as a potential treatment option for patients with COVID-19.

LBA78 A microsimulation model to assess the impact of SARS-CoV-2 on cancer outcomes, healthcare organization and economic burden

Background: SARS-CoV-2 pandemic has deeply modified healthcare seeking and services in Europe since February 2020 with delays in treatment delivery and changes in the standards of care. The organization of cancer centers (CC) has been transformed to minimize virus exposure in cancer patients (pts). Real-time assessment of the impact on cancer outcomes can optimize decision-making for future epidemic episodes. Methods: A discrete-event simulation (DES) model was developed to model individual pt pathways during the pandemic in a context of constrained medical resources. Cancer pt care is modeled based on pandemic-adapted guidelines for medical practice. Pt flow is derived from medico-administrative databases using time series methods to estimate the proportion of punctual / late visits and associated delay and to extrapolate future flows. Finally, the impact of modified care on survival is estimated using literature data. Results: From March to December 2020, based on data from Gustave Roussy CC in France (n= 4877 included pts), estimated overall treatment delay is <= 7 days in 86,6% of pts and 5,2% of pts have a delay higher than 2 months. More than 94% of this duration is delay in pt request for care, causing 99 pts to suffer a major prognosis change upon arrival. Delayed pt flows result in a highly time-variable use of medical resources, with important queues forecast for surgery care and chemotherapy. The handling of such queues will require intensified healthcare professionals effort. Projections show that, in the best-case scenario, ie without a 2nd pandemic wave, treatment delays and modifications will result in around 49 additional 5-year cancer-specific deaths (+ 2,25% of 5-year deaths), mainly in liver, sarcomas and head and neck cancer pts. Conclusions: In a resource-constrained context, optimization of the benefit-risk ratio between COVID-19 and cancer care is key. Simulations of individual projections from actual hospital data, show a 2.25% increase of the 5-year risk of death and that pandemic-related cancer burden is mainly due to patient-induced lateness in seeking care. Defining optimal strategies in terms of screening, monitoring and prioritization for care could minimize the impact of future pandemic episodes. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: A. Bardet: Advisory/Consultancy: Roche. M. Faron: Travel/Accommodation/Expenses: Ipsen;Travel/Accommodation/Expenses: Novartis;Travel/Accommodation/Expenses: Pfizer;Honoraria (self): HRA Pharma;Honoraria (self): Ipsen. I. Borget: Honoraria (self): Merck;Honoraria (self): CSL Berhing;Honoraria (self): Allergan;Honoraria (self): Novartis;Research grant/Funding (institution): BMS. S. Michiels: Advisory/Consultancy: IDDI;Advisory/Consultancy: Janssen Cilag;Honoraria (self), IDMC member: Hexal;Honoraria (self), IDMC member: Steba;Honoraria (self), IDMC member: IQVIA;Honoraria (self), IDMC member: Roche;Honoraria (self), IDMC member: Sensorion;Honoraria (self), IDMC member: Biophytis;Honoraria (self), IDMC member: Servier;Honoraria (self), IDMC member: Yuhan. F. Barlesi: Honoraria (self), further elements to be provided: AstraZeneca;Honoraria (self): Bayer;Honoraria (self): Bristol-Myers Squibb;Honoraria (self): Boehringer-Ingelheim;Honoraria (self): Eli Lilly Oncology;Honoraria (self): F.Hoffmann-La Roche Ltd;Honoraria (self): Novartis;Honoraria (self): Merck;Honoraria (self): MSD;Honoraria (self): Pierre Fabre;Honoraria (self): Pfizer;Honoraria (self): Takeda;Honoraria (institution): AbbVie;Honoraria (institution): Amgen;Honoraria (institution): AstraZeneca;Honoraria (institution): Bayer;Honoraria (institution): Bristol-Myers Squibb;Honoraria (institution): Boehringer-Ingelheim;Honoraria (institution): Eisai;Honoraria (institution): Eli Lilly Oncology;Honoraria (institution): F. Hoffmann-La Roche Ltd;Honoraria (institution): Genentech;Honoraria (institution): Ipsen;Honoraria (institution): Ignyta;Honoraria (institution): Innate Pharma;Honoraria (institu ion): Loxo;Honoraria (institution): Novartis;Honoraria (institution): MedImmune;Honoraria (institution): Merck, MSD, Pierre Fabre, Pfizer, Sanofi-Aventis, Takeda;Research grant/Funding (institution): AstraZeneca, BMS, Merck, Pierre Fabre, F. Hoffmann-La Roche Ltd. J. Bonastre: Honoraria (self): Bristol-Myers Squibb;Advisory/Consultancy: Bristol-Myers Squibb;Advisory/Consultancy: MSD;Advisory/Consultancy: PharmaMar (Inst);Advisory/Consultancy: Bristol-Myers Squibb (Inst);Advisory/Consultancy: Merck Serono;Travel/Accommodation/Expenses: Bristol-Myers Squibb. All other authors have declared no conflicts of interest.

Early lung ultrasound assessment for the prognosis of patients hospitalized for COVID-19 pneumonia. A pilot study.

OBJECTIVE: SARS CoV-2 is an epidemic viral infection that can cause mild to severe lung involvement. Newly apprehended knowledge on thoracic imaging abnormalities and the growing clinical experience on the evolution of this disease make the radiographic follow-up of hospitalized patients relevant. The value of consecutive bedside lung ultrasonography in the follow-up of hospitalized patients with SARS CoV-2 pneumonia and its correlation with other clinical and laboratory markers needs to be evaluated. METHODS: We assessed 39 patients [age: 64 y(60.1-68.7)] with confirmed SARS CoV-2 pneumonia. A total of 24 patients were hospitalized until the follow-up test, 9 were discharged early and 6 required a transfer to critical care unit. Two ultrasound scans of the lung were performed on day 1 and 4 of patients' hospitalization. Primary endpoint was the magnitude of association between a global lung ultrasound score (LUS) and clinical and laboratory markers. Secondary endpoint was the association between the evolution of LUS with the corresponded changes in clinical and laboratory outcomes during hospitalization period. RESULTS: LUS score on admission was higher among the deteriorating patients and significantly (P=0.038-0.0001) correlated (Spearman's rho) with the levels of C-reactive protein (0.58), lymphocytes (-0.33), SpO2 (-0.48) and oxygen supplementation (0.48) upon admission. The increase in LUS score between the two scans was significantly correlated (0.544, P=0.006) with longer hospital stay. CONCLUSION: Lung ultrasound assessment can be a useful as an imaging modality for SARS CoV-2 patients. Larger studies are needed to further investigate the predictive role of LUS in the duration and the outcome of the hospitalization of these patients.

Impact of Covid-19 on farming systems in Europe through the lens of resilience thinking.

Context: Resilience is the ability to deal with shocks and stresses, including the unknown and previously unimaginable, such as the Covid-19 crisis. Objective: This paper assesses (i) how different farming systems were exposed to the crisis, (ii) which resilience capacities were revealed and (iii) how resilience was enabled or constrained by the farming systems' social and institutional environment. Methods: The 11 farming systems included have been analysed since 2017. This allows a comparison of pre-Covid-19 findings and the Covid-19 crisis. Pre-Covid findings are from the SURE-Farm systematic sustainability and resilience assessment. For Covid-19 a special data collection was carried out during the early stage of lockdowns. Results and conclusions: Our case studies found limited impact of Covid-19 on the production and delivery of food and other agricultural products. This was due to either little exposure or the agile activation of robustness capacities of the farming systems in combination with an enabling institutional environment. Revealed capacities were mainly based on already existing connectedness among farmers and more broadly in value chains. Across cases, the experience of the crisis triggered reflexivity about the operation of the farming systems. Recurring topics were the need for shorter chains, more fairness towards farmers, and less dependence on migrant workers. However, actors in the farming systems and the enabling environment generally focused on the immediate issues and gave little real consideration to long-term implications and challenges. Hence, adaptive or transformative capacities were much less on display than coping capacities. The comparison with pre-Covid findings mostly showed similarities. If challenges, such as shortage of labour, already loomed before, they persisted during the crisis. Furthermore, the eminent role of resilience attributes was confirmed. In cases with high connectedness and diversity we found that these system characteristics contributed significantly to dealing with the crisis. Also the focus on coping capacities was already visible before the crisis. We are not sure yet whether the focus on short-term robustness just reflects the higher visibility and urgency of shocks compared to slow processes that undermine or threaten important system functions, or whether they betray an imbalance in resilience capacities at the expense of adaptability and transformability. Significance: Our analysis indicates that if transformations are required, e.g. to respond to concerns about transnational value chains and future pandemics from zoonosis, the transformative capacity of many farming systems needs to be actively enhanced through an enabling environment.

High dimensional profiling identifies specific immune types along the recovery trajectories of critically ill COVID19 patients.

The COVID-19 pandemic poses a major burden on healthcare and economic systems across the globe. Even though a majority of the population develops only minor symptoms upon SARS-CoV-2 infection, a significant number are hospitalized at intensive care units (ICU) requiring critical care. While insights into the early stages of the disease are rapidly expanding, the dynamic immunological processes occurring in critically ill patients throughout their recovery at ICU are far less understood. Here, we have analysed whole blood samples serially collected from 40 surviving COVID-19 patients throughout their recovery in ICU using high-dimensional cytometry by time-of-flight (CyTOF) and cytokine multiplexing. Based on the neutrophil-to-lymphocyte ratio (NLR), we defined four sequential immunotypes during recovery that correlated to various clinical parameters, including the level of respiratory support at concomitant sampling times. We identified classical monocytes as the first immune cell type to recover by restoration of HLA-DR-positivity and the reduction of immunosuppressive CD163 + monocytes, followed by the recovery of CD8 + and CD4 + T cell and non-classical monocyte populations. The identified immunotypes also correlated to aberrant cytokine and acute-phase reactant levels. Finally, integrative analysis of cytokines and immune cell profiles showed a shift from an initially dysregulated immune response to a more coordinated immunogenic interplay, highlighting the importance of longitudinal sampling to understand the pathophysiology underlying recovery from severe COVID-19.

Mapping the recovery of critically ill COVID19 patients by high-dimensional profiling identifies longitudinal blood immunotypes (preprint)

The COVID-19 pandemic poses a major burden on health-care and economic systems across the globe. Even though a majority of the population only develops minor symptoms upon SARS-CoV2 infection, a significant proportion are hospitalized at intensive care units (ICU) requiring critical care. While insights into the early stages of the disease are gradually expanding, the dynamic immunological processes occurring in critically ill patients throughout their recovery at ICU are far less understood. Here, we have analysed longitudinally collected, whole blood samples of 40 surviving COVID-19 patients during their recovery at ICU using high-dimensional cytometry by time-of-flight (CyTOF) and cytokine multiplexing. Based on the neutrophil to lymphocyte ratio (NLR), we defined 4 sequential immunotypes during recovery that correlated to various clinical parameters, including the level of respiratory support at concomitant sampling times. We also identified classical monocytes as the first immune cell type to recover by restoring HLA-DR-positivity and by reducing the immunosuppressive CD163+ monocyte population, followed by the recovery of CD8+ and CD4+ T cell, and mDC populations. The identified immunotypes also correlated to aberrant cytokine and acute-phase reactant levels. Finally, integrative analysis of cytokines and immune cell profiles showed a shift from an initially dysregulated immune response to a more coordinated immunogenic interplay, highlighting the importance of longitudinal sampling to understand the pathophysiology underlying recovery from severe COVID-19.

Keeping exhausted T-cells in check in COVID-19

Background: Clinical data suggest an aggravated COVID-19 disease course in cancer patients treated with immune checkpoint inhibitors (ICI). European guidelines advise to defer ICI therapy until complete resolution of COVID-19. However, mechanistic insight into how ICI impacts COVID-19 immunopathology is absent. Methods: We performed single-cell RNA- and T-Cell Receptor-sequencing (TCR-seq) on bronchoalveolar lavage fluid of COVID-19 pneumonia (n=19) and non-COVID pneumonia (n=10), and co-analyzed CD8+ T-cells with publicly available tumor-infiltrating T-cell data of treatment-naïve and ICI-treated patients (Sade-Feldman, Cell, 2018;Lambrechts, Nat Med, 2018). Cell lineages were determined by trajectory inference (Slingshot, Monocle v2) and stratified per condition. Pathogen- or tumor-directed T-cells were defined based on clonal selection (Zhang, Nature, 2018). To identify ICI responsive cells, we calculated a score derived from a validated gene set denoting ICI reactivity (Okamura, J. Autoimmun, 2019). Results: We identified 3 CD8+ T-cell lineages, with ‘Naïve’ T-cells transitioning into ‘Effector Memory’ cells and then branching into either ‘Recently Activated Effector Memory (TEMRA)’, ‘Exhausted (TEX)’ or ‘Resident Memory (TRM)’ T-cells. In COVID-19, clonal expansion indicating a SARS-CoV-2 antigen-specific T-cell response, was mainly observed in the highly cytotoxic ‘TEMRA’ lineage. In contrast, tumor-specific T-cells were found in the ‘TEX’ lineage. Of importance, the ICI responsiveness score was significantly higher in the non-pathogen-directed ‘TRM’ and ‘TEX’ cells in COVID-19. In cancer, ‘TEX’ cells were shown to be ICI responsive as expected. [Formula presented] Conclusions: We are the first to provide a mechanistic rationale for an aggravated COVID-19 disease course in ICI-treated patients. Whereas ICI reactivates tumor-directed ‘exhausted’ T-cells in cancer, it preferentially potentiates non-pathogen-directed T-cells in COVID-19, thereby contributing to lung damage without boosting the antiviral immune response. Clinical trial identification: In-depth Immunological Investigation of COVID-19 (COntAGIouS). - Clinical Trial identifier: NCT04327570. - Ethical approval obtained by the Ethics Committee of University Hospitals - KU Leuven. File number S63881. Legal entity responsible for the study: University Hospitals - KU Leuven. Funding: Kom op tegen Kanker (Stand up to Cancer). Disclosure: All authors have declared no conflicts of interest.