Mid-density Gene Expression profiling of SARSCOV-2 infected samples using Applied BiosystemsTM TaqManTM Flexible Array Panels

Background/Objectives: The broad spectrum of clinical manifestations from SARS-COV-2 infection and observed risk factors for severe disease highlight the importance of understanding molecular mechanisms underlying SARS-CoV-2 associated disease pathogenesis. Research studies have identified a large number of host proteins that play roles in viral entry, innate immune response, or immune signalling during infection. The ability to interrogate subsets of these genes simultaneously within SARSCOV-2 infected samples is critical to understanding how their expression contribute to phenotypic variability of the disease caused by the pathogen. Method(s): 30 Nasopharyngeal swab were obtained and included SARS-CoV-2 infected and control samples. RNA was extracted, reverse transcribed and loaded onto flexible TaqMan array panels designed specifically for targeting the most cited genes related to SARS-COV-2 entry and restriction factors as well as cytokines, chemokines, and growth factors involved in the pathogenesis. Result(s): Our data indicated that not only were the levels of several of these host factors differentially modulated between the two study groups, but also that SARS-CoV-2 infected subjects presented with greater frequency of several important inflammatory cytokines and chemokines such as CCL2, CCL3, IFNG, entry receptors such as ACE2, TMRPS11A, and host restriction factors including LY6E and ZBP1. Conclusion(s): TaqMan array plates provide a fast, midthroughput solution to determine the levels of several virus and host-associated factors in various cell types and add to our understanding of how the pathogenesis may vary depending on gender, age, infection site etc.

Inflammatory Pathways Associated with Covid-19 Severity Differ Based on Obesity Status

Background: Severe COVID-19 and obesity are characterized by higher inflammation. We aimed to examine early inflammatory patterns in people with (Ob) and without (NOb) obesity and COVID-19 and how they relate to COVID-19 disease severity Methods: Ob (BMI >30 Kg/m2) and NOb with COVID-19 matched for age, sex and WHO disease severity provided blood early after diagnosis. Immunoassays measured 57 plasma biomarkers reflecting innate immune and endothelial activation, systemic inflammation, coagulation, metabolism and microbial translocation (Fig 1). Between-group differences were assessed by Mann- Whitney. Associations between subsequent maximal COVID-19 severity (mild vs moderate/severe/critical) and biomarkers were explored by logistic regression adjusted for age, sex, hypertension (HTN) and diabetes (DM). Data are median pg/mL [IQR] or n [%] unless stated Results: Of 100 subjects (50 Ob and 50 Nob) presenting between April 2020 and March 2021, characteristics (Ob vs Nob) included: age 65 [23-91] vs 65 [21-95];female sex 27 (48%) vs 28 (56%);BMI 33.7 [30.0-71.8] vs 23.3 [15.3-25.9];disease severity mild 22 [48%] vs 23 [46%], moderate 15 [30%] vs 13 [26%], severe 6 [12%] vs 7 [14%];HTN 30 (60%) vs 17 (34%);DM 19 [38%] vs 6 [12%];days from symptom onset 7 [2-17] vs 8 [1-15];vaccinated 3 (6%) vs 0 (0%). Compared to NOb, Ob had higher IFN-alpha (1.8 [0.6;11] vs 0.9 [0.1;4.7]), CRP (10 mAU/mL [9.6;10.2] vs 9.7 [7.2;10]), IL-1RA (197 [122;399] vs 138 [88;253]), IL-4 (288 AU/mL [161;424] vs 205 [82;333]), vWF (252 [166;383] vs 163 [96;318]), Zonulin (114 ng/mL [77;131] vs 57 [18;106]), Resistin (956 [569;1153] vs 727 [712;1525]), Leptin (3482 [1513;5738] vs 848 [249;2114]), and lower Adiponectin (1.12 mg/L [0.09;1.5] vs 1.5 [1.18;1.93]), all p< 0.05. In both groups higher, proinflammatory IL-18 and lower levels of antiinflammatory CCL22 and IL-5 were associated with higher odds of disease severity, and lower E-selectin with higher disease severity only in Ob. However, in NOb higher type 3 interferons (IL-28A), macrophage activation (sCD163, CCL3) and vascular inflammation markers (ICAM-1, VCAM-1), along with higher S100B, GM-CSF and leptin were also associated with disease severity, a pattern not observed in Ob (Fig 1) Conclusion(s): Although Ob had higher overall levels of inflammation than NOb, few biomarkers predicted subsequent COVID-19 severity in Ob. These differential inflammatory patterns suggest dysregulated immune responses in Ob with COVID-19. (Figure Presented).

Cytokine Profile in Different Post-Covid-19 Condition Phenotypes

Background: At least 10% of SARS-CoV-2 infected patients suffer from persistent symptoms for >12 weeks, known as post-COVID-19 condition (PCC) or Long Covid. Reported symptomatology is diverse with >200 physical and neurological debilitating symptoms. Here, we analyzed pro-inflammatory cytokine levels as a potential mechanism underlying persistent symptomatology. Method(s): Clinical data and samples used belong to the KING cohort extension, which includes clinically well characterized PCC (N=358, 59 persistent symptoms evaluated), COVID-19 recovered and uninfected subjects. We used Gower distances to calculate symptom's similarity between PCC and Ward's hierarchical clustering method to identify different symptom patterns among PCC patients. Cytokine levels of randomly selected PCC, recovered and uninfected subjects (N=193) were measured on plasma samples collected >6 months after acute infection using the 30-Plex Panel for Luminex. Mann- Whitney t-test was used to compare PCC vs recovered groups and Kruskal-Wallis t-test for >2 groups comparisons (PCC vs recovered vs Uninfected and within PCC clusters). FDR correction was applied for statistical significance (p-adj). Result(s): Hierarchical clustering identified 5 different PCC clusters according to their symptomatology, where PCC3 and PCC5 clusters showed higher prevalence of women ( >80%) and more persistent symptoms, while acute COVID-19 was mild in >80% of the patients. We selected 91 PCC (belonging to each cluster), 57 recovered and 45 uninfected subjects for cytokine profiling (Table 1). 13 soluble markers were significantly elevated (IL-1beta, Eotaxin, MIP-1beta, MCP-1, IL-15, IL-5, HGF, IFN-alpha, IL-1RA, IL-7, MIG, IL-4 and IL-8) in PCC and recovered groups compared to uninfected subjects (all p-adj< 0.04). In addition, PCC subjects tended towards higher levels of IL-1RA compared to recovered group (padj= 0.071). Within PCC clusters, FGF-basic and RANTES were elevated while IL-2 and MIG were decreased in PCC3 and PCC5 compared to the other PCC clusters (all p-adj< 0.04). TNF-alpha, IP-10, G-CSF and MIP-1alpha were decreased in PCC3 and PCC5 not reaching statistical significance (all p-adj=0.07). Conclusion(s): Some cytokines remained altered in all SARS-CoV-2 infected subjects independently of persistent symptoms after 6 months from acute infection. Differences between PCC and recovered individuals are limited after correction. Importantly, PCC cytokine profiles showed differences between clusters, which suggests different PCC subsyndromes with distinct etiology. Subjects Characteristics (Table Presented).

Continuous renal replacement therapy with oXiris filter in critically ill COVID-19 patients

Introduction: COVID-19 causes a major inflammatory response, which may progress to shock and multiple organ failure. We explored whether continuous renal replacement therapy (CRRT) using adsorption membrane (oXiris) could reduce the inflammatory response in critically ill COVID-19 patients with acute renal failure (ARF) [1, 2]. Method(s): Case-control study including 24 critically ill COVID-2019 patients requiring RRT using an oXiris filter. We measured cytokines before and during treatment as well as relevant clinical endpoints. The control group was selected among COVID-19 patients included into our ongoing RECORDS trial (NCT04280497) who received RRT without adsorbing filters. Result(s): 24 severe COVID-19 patients, admitted to the intensive care unit (ICU) and treated with CRRT using the oXiris filter between March and April 2020 (20 males and 4 females);median age 67. The average time from COVID-19 symptoms to initiation of oXiris treatment was 18 +/- 7 days, and from ICU admission to initiation of oXiris treatment 9.5 +/- 7.8 days and from ARF to oXiris treatment was 3 +/- 5 days. The average length of treatment was 152.8 +/- 92.3 h. Treatment was associated with cytokine decreases for IL-1beta (p = 0.00022), MCP-1 (p = 0.03), and MIP-1 alpha (p = 0.03). The SOFA scores also showed a reduction over 48 h of therapy without reaching statistical significance. Our study found no significant effect of hemodynamic status. The average ICU stay length was 14 +/- 5 days and the mortality rate was 79% in the Oxiris group. We compared the mortality across the two matched groups, there was no evidence of any difference in mortality (Fig. 1). Conclusion(s): In our study, CRRT using the oXiris filter seemed to effectively remove IL-1 beta, MCP-1, and MIP-1 alpha in COVID-19 patients. These exploratory results should be confirmed in a randomized controlled study.

Inflammatory and Blood-Brain Barrier Markers after Covid-19 among People with Hiv

Background: COVID-19, the disease caused by SARS-CoV-2, has resulted in devastating morbidity and mortality worldwide. Alarming evidence indicates that long-term adverse outcomes of COVID-19 can affect all major systems of the body, including the immune, respiratory, cardiovascular, and neurological systems. While acute COVID-19 pathology does not appear to be markedly different by HIV status, long-term outcomes of COVID-19 in People with HIV (PWH) are unknown and require further investigation. This study evaluates the inflammatory profile longitudinally up to three months after COVID-19. In addition, markers of the blood-brain barrier (BBB) integrity and vascular dysfunction were also evaluated. Method(s): Plasma samples were collected from 15 males and 6 females with COVID-19 and HIV infection (COVID+/HIV+) and 9 males and 14 females with COVID-19 without HIV infection (COVID+/HIV-) between March 2020 and March 2021. Baseline samples were obtained approx. 10 days after COVID-19 diagnosis (T=0) and three months after (T=3). Mean age group for COVID+/HIV-was 45.4+/-17.8 years for males and 39.7+/-15.3 for females and for COVID+/HIV+ was 52.1+/-12.3 for males and 48.7+/-1 for females (N=15 and 6, respectively). 27 inflammatory molecules were measured by Bio-Plex Multiplex Immunoassay (Bio-Rad) and two markers of BBB and vascular dysfunction (soluble ICAM1 and S100beta) by ELISA. Result(s): Out of 27 inflammatory analytes, 20 had detectable signals. Eotaxin (CCL11) and G-CSF levels were differentially upregulated in the COVID+/HIV+ group as compared to the COVID+/HIV-group in both time point studied (Table 1). IFN-g showed sustained increased levels at T=3 in the COVID+/HIV+ group, whereas there was a significant reduction over time in the COVID+/HIV-group. At T3, inflammatory markers (IL-4, IL-8, IL-13, basic FGF, TNF-alpha, MIP-1alpha, and CCL2) either decreased or remained unchanged in both groups. In contrast, the markers of the BBB disruption and vascular dysfunction, such as S100beta and soluble ICAM-1 increased in the COVID+/HIV+ group, suggesting long-term progressive BBB and vascular alterations. Conclusion(s): HIV-1 may potentiate long COVID-19-induced neuropathology, with progressive BBB breakdown and sustained increase in eotaxin-1 and G-CSF. Plasma inflammatory markers in COVID-19 patients with or without HIV-1 co-infection.

Potential Predictors for Disease Severity in Hospitalised COVID-19 Patients

Background and Objectives Several studies have shown that SARS-CoV-2 can induce a cytokine release storm which is a major cause of disease severity and death. Therefore, cytokine levels in the serum may predict disease severity and survival in patients with COVID-19. Methods We included 88 COVID-19 patients who were hospitalised at the Division of Pulmonology of the Vienna General Hospital between January and May 2021 in this observational trial. Blood samples for serum peptide measurements were drawn at the time closest to hospitalisation, at day 5, 9 and 13( +/- 1). We correlated the type of ventilation (nasal oxygen therapy, high flow nasal canula, non-invasive ventilation or mechanical ventilation), occurrence of consolidations on chest X-ray or if available HRCT and the level of care (general ward, IMCU or ICU) with serum peptide values. We assessed the concentration of cytokines (IL-1a, IL-1b, IL-1RA, IL-6, L-7, L-10, IFN- gamma and TNF-alpha), chemokines (CCL-3, CCL-4 and CCL-7) and growth factors (G-CSF, GM-CSF and VEGF). Results Patients were 68 years of age (median) and stayed in hospital between 5-171 days. The peak inspiratory pressure in patients receiving non-invasive ventilation or mechanical ventilation was significantly associated with IL-1RA, G-CSF and IFN-gamma and the fraction of inspired oxygen in patients receiving highflow nasal canula oxygen therapy was significantly associated with IL-6, IL-7, IFN-gamma, and CCL-7. Results are shown in Table 1. No investigated cytokine correlated with the type of ventilation, occurrence of consolidations on imaging and in-hospital mortality. Conclusions In conclusion, concentrations of IL-1RA, G-CSF, IL-6, IL-7, IFN-gamma, and CCL-7 were associated with more severe disease progression in hospitalised COVID-19 patients.

Understanding kidney injury in covid-19, a pressing priority

The 2019 novel coronavirus disease (COVID-19) is a newly defined infectious and highly contagious acute disease caused by the severe acute respiratory syndrome coronavirus 2 ( (SARS-CoV-2). COVID-19 is mainly characterized by an acute respiratory disease however it can also affect multiple other organ systems such as the kidney, gastrointestinal tract, heart, vascular system, and the central nervous system. Kidney involvement is frequent in patients with COVID-19 and this review aims to explore the available data on kidney and COVID-19. In conclusion, COVID-19 infection can affect renal function and may cause acute kidney injury (AKI), due to several mechanisms that need to be fully elucidated. As only supportive management strategies are available for treating AKI in COVID-19, it is necessary to identify and preserve renal function during SARS-CoV-2 infection.Copyright © 2021 The Author(s).

Predictive Value of Specific Cytokines for Lethal Covid-19 Outcome

In our study, we aimed to evaluate the significance of specific cytokines in blood plasma as predictive markers of COVID-associated mortality. Materials and methods. In plasma samples of 29 patients with PCR-confirmed COVID-19 we measured the concentrations of 47 molecules. These molecules included: interleukins and selected pro-inflammatory cytokines (IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A/CTLA8, IL-17-E/IL-25, IL-17F, IL-18, IL-22, IL-27, IFNalpha2, IFNgamma, TNFalpha, TNFbeta/Lymphotoxin-alpha(LTA));chemokines (CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL7/MCP-3, CCL11/Eotaxin, CCL22/MDC, CXCL1/GROalpha, CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, CX3CL1/Fractalkine);anti-inflammatory cytokines (IL-1Ra, IL-10);growth factors (EGF, FGF-2/FGF-basic, Flt-3 Ligand, G-CSF, M-CSF, GM-CSF, PDGF-AA, PDGFAB/BB, TGFalpha, VEGF-A);and sCD40L. We used multiplex analysis based on xMAP technology (Luminex, USA) using Luminex MagPix. As controls, we used plasma samples of 20 healthy individuals. Based on the results, we applied Receiver Operating Characteristic (ROC) analysis and Area Under Curve (AUC) values to compare two different predictive tests and to choose the optimal division point for disease outcome (survivors/non-survivors). To find optimal biomarker combinations, we as used cytokines concentrations as dependent variables to grow a regression tree using JMP 16 Software.Results. Out of 47 studied cytokines/chemokines/growth factors, we picked four pro-inflammatory cytokines as having high significance in evaluation of COVID-19 outcome: IL-6, IL-8, IL-15, and IL-18. Based on the results received, we assume that the highest significance in terms of predicting the outcome of acute COVID-19 belongs to IL-6 and IL-18. Conclusion. Analyzing concentrations of IL-6 and IL-18 before administering treatment may prove valuable in terms of outcome prognosis. Copyright © Arsentieva N.A. et al., 2022.

Relation between macrophage inflammatory protein-1 and intercellular adhesion molecule-1 and computed tomography findings in critically-ill saudi covid-19 patients.

BACKGROUND: Several, clinical and biochemical factors were suggested as risk factors for more severe forms of Covid-19. Macrophage inflammatory protein-1 alpha (MIP-1α, CCL3) is a chemokine mainly involved in cell adhesion and migration. Intracellular adhesion molecule 1 (ICAM-1) is an inducible cell adhesion molecule involved in multiple immune processes. The present study aimed to assess the relationship between baseline serum MIP-1α and ICAM-1 level in critically-ill Covid-19 patients and the severity of computed tomography (CT) findings. METHODS: The study included 100 consecutive critically-ill patients with Covid-19 infection. Diagnosis of infection was established on the basis of RT-PCR tests. Serum MIP-1α and ICAM-1 levels were assessed using commercially available ELISA kits. All patients were subjected to a high-resolution computed tomography assessment. RESULTS: According to the computed tomography severity score, patients were classified into those with moderate/severe (n=49) and mild (n = 51) pulmonary involvement. Severe involvement was associated with significantly higher MIP-1α and ICAM-1 level. Correlation analysis identified significant positive correlations between MIP-1α and age, D-dimer, IL6, in contrast, there was an inverse correlation with INF-alpha. Additionally, ICAM-1 showed significant positive correlations with age, D-Dimer,- TNF-α, IL6,while an inverse correlation with INF-alpha was observed. CONCLUSIONS: MIP-1α and ICAM-1 level are related to CT radiological severity in Covid-19 patients. Moreover, these markers are well-correlated with other inflammatory markers suggesting that they can be used as reliable prognostic markers in Covid-19 patients.

Higher Proinflammatory Cytokines Are Associated with Increased Antibody Titer After a Third Dose of SARS-CoV-2 Vaccine in Solid Organ Transplant Recipients

Purpose: Solid organ transplant recipients (SOTRs) are at increased risk for severe COVID-19 and exhibit lower antibody responses to SARS-CoV-2 vaccines. This study aimed to determine if pre-vaccination cytokine levels are associated with antibody response to SARS-CoV-2 vaccination. Method(s): A cross-sectional study was performed among 58 SOTRs before and after two-dose mRNA vaccine series, 35 additional SOTRs before and after a third vaccine dose, with comparison to 16 healthy controls (HCs). Anti-spike antibody was assessed using the IgG Euroimmun ELISA. Electrochemiluminescence detectionbased multiplexed sandwich immunoassays were used to quantify plasma cytokine and chemokine concentrations (n=20 analytes). Concentrations between SOTRs and HCs, stratified by ultimate antibody response to the vaccine, were compared using Wilcoxon-rank-sum test with false discovery rates (FDR) computed to correct for multiple comparisons. Result(s): In the study population, 100% of HCs, 59% of SOTRs after two doses and 63% of SOTRs after three doses had a detectable antibody response. Multiple baseline cytokines were elevated in SOTRs versus HCs. There was no significant difference in cytokine levels between SOTRs with high vs low-titer antibodies after two doses of vaccine. However, as compared to poor antibody responders, SOTRs who went on to develop a high-titer antibody response to a third dose of vaccine had significantly higher pre-third dose levels of several innate immune cytokines including IL-17, IL-2Ra, IL-6, IP-10, MIP-1alpha, and TNF-alpha (FDR <0.05). Conclusion(s): A specific inflammatory profile or immune state may identify which SOTRs are likely to develop stronger sero-response and possible protection after a third dose of SARS-CoV-2 vaccine.

Correlation of COVID-19 Biomarkers with Disease Severity: A Retrospective Study

Introduction: Many viruses through aerosols, droplets, and droplet nuclei utilize the respiratory passages to establish not only localized respiratory tract infections but also systemic disease. The coronaviruses (CoV) are no exception. The two most common illnesses that occurred in the recent past were severe acute respiratory syndrome (SARS, 2003) and the Middle East respiratory syndrome (MERS, 2012).1 The current pandemic, which broke out in late December 2019, has been a major threat to global public health due to significant morbidity and mortality, akin to snapping of Thanos' fingers. The novel coronavirus was initially named the 2019-novel CoV (2019-nCoV), but because of nearly 80% genetic homology to SARS-CoV, the Coronavirus Study Group of International Committee rechristened this virus as SARS-CoV-2.1 The disease was named coronavirus disease 2019 (COVID-19) on January 12, 2020, by the World Health Organization (WHO).2 According to the Advisory Committee on dangerous pathogens UK, COVID-19 is assigned as a hazardous group-3 organism, meaning that it can cause severe human disease.3 The novel coronavirus was named the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, 2019-nCoV) due to its high homology (∼80%) to SARS-CoV, which caused acute respiratory distress syndrome (ARDS) and high mortality during 2002-2003.4 The outbreak of SARS-CoV-2 was considered to have originally started via a zoonotic transmission associated with the seafood market in Wuhan, China. Later it was recognized that human-to-human transmission played a major role in the subsequent outbreak.5 The most common clinical manifestations of COVID-19 include fever, cough, dyspnea, fatigue, and myalgia. A few patients have developed severe pneumonia and they may present with acute respiratory distress syndrome (ARDS), extrapulmonary organ dysfunction, or even death. SARS-CoV-2 virus primarily affects the respiratory system, although other organ systems are also involved. Lower respiratory tract infection-related symptoms including fever, dry cough, and dyspnea were reported in the initial case series.6 In addition, headache, dizziness, generalized weakness, vomiting, and diarrhea were observed.7 It is now widely recognized that respiratory symptoms of COVID-19 are extremely heterogeneous, ranging from minimal symptoms to significant hypoxia with ARDS. The heterogeneous disease course of COVID-19 is unpredictable with most patients experiencing mild self-limiting symptoms. However, up to 30% require hospitalisation, and up to 17% of these require intensive care support for acute respiratory distress syndrome (ARDS), hyperinflammation, and multiorgan failure. 8-10 A cytokine storm in patients with severe disease was identified in the early reports of Wuhan patients and is intrinsic to disease pathology. In this cohort, elevated plasma interleukin (IL)-2, IL-7, IL-10, granulocyte colony-stimulating factor (GCSF), interferon γ-induced protein 10 (IP10, monocyte chemoattractant protein-1 (MCP1), macrophage inflammatory protein 1-alpha (MIP1A), and tumor necrosis factor-alpha (TNF-α) levels in ICU patients were identified. 6 Studies have shown that severe or fatal cases of COVID-19 disease are associated with an elevated white cell count, blood urea nitrogen, creatinine, markers of liver and kidney function, C-reactive protein (CRP), interleukin-6 (IL-6), lower lymphocyte (<1000/μL) and platelet counts (<100 × 109/L) as well as albumin levels compared with milder cases in which survival is the outcome. Subsequent studies have implicated IL-6 as a valuable predictor of adverse clinical outcome and a potential therapeutic target.11,12 One or more clinical and wet biomarkers may enable early identification of high-risk cases, assisting disease stratification and effective use of limited specialist resources. Age is a strong risk factor for severe illness, complications, and death.13,14 Patients with no underlying medical comorbid conditions have an overall case fatality rate of <1%. Case fatality is higher for patients with comorbidit es. The severe cases are associated with elevated levels of inflammatory biomarkers such as serum lactate dehydrogenase, creatine kinase, C-reactive protein (CRP), d-dimer, procalcitonin, and ferritin.15 Since laboratory medicine has always supported clinical decision-making in various infectious diseases, it is important to assess the ability of laboratory-derived biomarkers to facilitate risk stratification of COVID-19 disease. This study will comprehensively explore clinical disease features and routine laboratory tests associated with COVID-19 disease and its complications, to address their association with disease severity and outcome. Hence, the present retrospective study will be done at our tertiary care centre to assess the association between different laboratory biomarkers and disease severity and outcomes in COVID-19 patients. Aims and objectives: Clinical correlation of biomarkers and disease severity in COVID-19 patients-a retrospective study. Review of Literature: Xia et al.16 in 2020 defined disease stages and identified stages' determining factors are instructive for the definition of standards for home quarantine. The authors demonstrated pulmonary involvement on a chest CT scan in 97.9% of cases. It took 16.81 ± 8.54 (3-49) days from the appearance of the first symptom until 274 patients tested virus-negative in naso- and oropharyngeal (NP) swabs, blood, urine, and stool, and 234 (83%) patients were asymptomatic for 9.09 ± 7.82 (1-44) days. Subsequently, 131 patients were discharged. One hundred and sixty-nine remained in the hospital;these patients tested virus-free and were clinically asymptomatic because of widespread persisting or increasing pulmonary infiltrates. Hospitalization took 16.24 ± 7.57 (2-47) days;the time interval from the first symptom to discharge was 21.37 ± 7.85 (3-52) days. The authors concluded that with an asymptomatic phase, disease courses are unexpectedly long until the stage of virus negativity. NP swabs are not reliable in the later stages of COVID-19. Pneumonia outlasts virus-positive tests if sputum is not acquired. Imminent pulmonary fibrosis in high-risk groups demands follow-up examinations. Investigation of promising antiviral agents should heed the specific needs of mild and moderate COVID-19 patients. Keddie et al.17 in 2020 investigated the routine laboratory tests and cytokines implicated in COVID-19 for their potential application as biomarkers of disease severity, respiratory failure, and need for higher-level care. The authors found CRP, IL-6, IL-10, and LDH were most strongly correlated with the WHO ordinal scale of illness severity, the fraction of inspired oxygen delivery, radiological evidence of ARDS, and level of respiratory support. IL-6 levels of ≥3.27 pg/mL provide a sensitivity of 0.87 and specificity of 0.64 for a requirement of ventilation, and a CRP of ≥37 mg/L of 0.91 and 0.66. The authors concluded that reliable stratification of highrisk cases has significant implications on patient triage, resource management, and potentially the initiation of novel therapies in severe patients. Malik et al.18 in 2020 in a systematic review and meta-analysis assessed the role of biomarkers in evaluating the severity of disease and appropriate allocation of resources. Studies having biomarkers, including lymphocyte, platelets, d-dimer, lactate dehydrogenase (LDH), C-reactive protein (CRP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, procalcitonin (PCT), and creatine kinase (CK), and describing outcomes were selected with the consensus of three independent reviewers. The authors found lymphopenia, thrombocytopenia, elevated d-dimer, elevated CRP, elevated PCT, elevated CK, elevated AST, elevated ALT, elevated creatinine, and LDH were independently associated with a higher risk of poor outcomes. The authors concluded a significant association between lymphopenia, thrombocytopenia, and elevated levels of CRP, PCT, LDH, d-dimer, and COVID-19 severity. The results have the potential to be used as an early biomarker to impro e the management of COVID-19 patients, by identification of high-risk patients and appropriate allocation of healthcare resources in the pandemic. Tjendra et al.19 in 2020 assessed specific laboratory parameters and summarized the currently available literature on the predictive role of various biomarkers in COVID-19 patients.

Interferon-mediated Responses to Human Metapneumovirus

Background: Acute respiratory infection (ARI) is the leading infectious cause of pediatric death worldwide, comprising 15% of all deaths in children under 5 years old. Human metapneumovirus (HMPV) is a primary cause of ARI, and accounts for a major portion of ARI-related hospitalizations in infants and young children. Although nearly every person is infected with HMPV during early childhood, re-infections occur often, highlighting the difficulty in building long-term immunity. There are no approved vaccines or antiviral therapies. Early host responses to HMPV are poorly characterized, and further understanding could identify important antiviral pathways and potential therapeutic targets. Type I (IFN-α/β) and III interferons (IFN-λ) display antiviral activity against numerous respiratory viruses and are currently being investigated for therapeutic use in several respiratory infections including SARS-CoV-2. However, their roles in HMPV infection remain largely unknown. Our laboratory has previously shown that type I IFN is critical for HMPV pathogenesis, as loss of IFN-α/β signaling reduces lung inflammation and lessens HMPV disease severity in mice. Here, we describe distinct antiviral roles for type I and III IFNs during HMPV infection using an established mouse model. Methods: In vivo studies were conducted using mice lacking either the IFN-α/ β receptor (IFNAR-/-) or IFN-λ receptor (IFNLR-/-). Early immune responses to HMPV strains TN/94-49 and C2-202 were assessed by clinical disease scoring, plaque assay, Luminex immunoassay, and spectral cytometry of mouse lung samples. In vitro studies were performed using CMT 64-61 mouse bronchial epithelial cells. Responses to TN/94-49 and C2-202 were measured by qPCR, plaque assay, and Luminex immunoassay of cell lysates and supernatants. Results: IFNAR-/- mice exhibited lower clinical disease scores, reduced lung levels of inflammatory cytokines IL6, MIP-1α, and MCP-1, and decreased numbers of lung interstitial macrophages during HMPV infection, highlighting their critical role in HMPV immune-mediated pathogenesis. IFNLR-/- mice with intact IFNAR showed moderate clinical disease, higher lung levels of inflammatory cytokines IL-6, MCP-1, and IFN-γ, and increased lung interstitial macrophage recruitment. A reduction in HMPV disease was also recapitulated by IFNAR-neutralizing antibody treatment of IFNLR-/- mice. Interestingly, IFNLR-/- showed higher HMPV viral titers, while IFNAR-/- mice showed no differences or slightly lower viral titers, compared to wild-type mice. Moreover, IFN-λ pre-treatment of infected CMT 64-61 cells reduced HMPV viral titers and decreased supernatant levels of inflammatory cytokines IL-6, IL-1β, TNFα, and MCP-1. Conclusion: These findings suggest that type I IFN is necessary for HMPV pathogenesis, while type III IFN is critical for limiting HMPV replication in the lungs but does not contribute to HMPV inflammatory disease. This work uncovers key functional differences between type I and III IFNs during HMPV infection, an important feature of innate immune responses to HMPV that may be utilized to inform treatment.

TREK-1 Potassium Channel Activation Protects Against Influenza-A Induced Lung Injury

Introduction and Rationale: No targeted therapies exist that improve the outcomes of patients with Acute Respiratory Distress Syndrome (ARDS), in part to the multifactorial etiology of this devastating disease. Infectious agents remain the most common initiating insults, and besides SARS-CoV-2, Influenza-A virus (IAV) is responsible for more ARDS cases and deaths than any other agent. In fact, IAV increases the risk of mortality in ARDS patients three-fold, and accounts for almost half of all ARDS deaths. We recently identified TREK-1 potassium channels on epithelial cells as important regulators of alveolar inflammation and barrier function, two hallmarks of ARDS, and found that pharmacological activation of TREK-1 protects against hyperoxia-induced lung injury. However, whether TREK-1 channels convey similar protection in a clinically more relevant IAVinduced lung injury model, remains unknown. Methods: We infected adult C57BL/6 wildtype mice intra-tracheally (i.t.) with IAV (PR8 strain;TCID50 400), followed by once-daily i.t. injections (days 5, 6 and 7 post-IAV) with the novel TREK-1 activating compounds ML335 (60mcg/kg), BL1249 (100mcg/kg), or a vehicle control, to create a clinically-relevant treatment model. To evaluate the role of epithelial cells in this model, we infected primary human alveolar epithelial cells (HAEC) with IAV (0.01 pfu) for 24 hours. Endpoint analysis consistent in quantification of quasi-static lung compliance;BAL fluid total protein, cell counts, and ROS concentrations;cytokine levels in BAL fluid and cell supernatants;and HAEC viability (XTT assay). In addition, we measured alterations in epithelial potassium currents (fluorometric FLIPR assays) and in IAV-induced signaling cascades (real-time PCR) following IAV infection and treatment with our TREK-1 activators. Results: Oncedaily treatment of mice with the TREK-1 activating compounds ML335 or BL1249 following IAV infection improved lung compliance, and BAL fluid total protein levels, cell counts, IL-6, CXCL-10, MIP-1alpha, and TNF-alpha concentrations, but not ROS, CCL-2 or IL-10 levels. In HAEC, TREK-1 activation improved IAV-induced IL-6, CXCL-10, and CCL-2 levels, while MIP-1alpha, TNF-alpha and IL-10 levels remained unchanged. XTT assays confirmed that in our model IAV infection did not cause significant cell death. Interestingly, IAV infection decreased HAEC potassium currents, which could be counteracted by TREK-1 activation and cell hyperpolarization. Finally, TREK-1 activationmediated cell hyperpolarization inhibited TLR4- and TNFSF13-mediated downstream signaling in IAV-infected HAEC, whereas NFkB, RIG1, TLR3, and TLR7 signaling was not affected. Conclusions: TREK-1 potassium channel activation may represent a novel approach to protect against IAV-induced acute lung injury.

GALECTIN-9 PROTECTS HUMANIZED ACE2-IMMUNOCOMPETENT MICE from SARS-CoV-2 INFECTION

Background: Coronavirus disease 2019 (COVID19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) remains a global health emergency even with effective vaccines and limited FDA-approved therapies. To limit mortality and morbidity across the spectrum of disease, the need for therapeutics remains critical. Galectin9 (gal9) is a beta-galactoside binding protein that modulates cell-cell and cell-matrix interactions. In response to SARS-CoV2 infection, it has been shown that circulating gal9 levels are elevated in patient sera with moderate to severe disease. Additionally, it has been reported that gal9 unexpectedly may competitively bind the host ACE2 receptor, potentially impeding viral entry. Therefore, we hypothesized that early recombinant gal-9 treatment post infection may prevent binding of the virus to susceptible host cells resulting in decreased severity of SARS-CoV2-associated disease. Methods: To determine the therapeutic potential of gal9 for treating COVID19, we infected K18-hACE2 transgenic mice intranasally with 104 particle forming units (PFU) of SARS-CoV2. 6 hours post infection (hpi), mice were treated with a single dose of 30 ug of recombinant human gal9 (rhgal9) or PBS intraperitoneally and subsequently monitored 12 days for morbidity. Subgroups of mice were humanely euthanized at 2 and 5 days pi (dpi) for viral plaque assay, flow cytometry, and protein analysis from lung tissue and bronchial alveolar lavage (BAL). Results: We found that mice treated with rhgal9 during the acute phase of infection exhibit improved survival compared to PBS treated animals (25%, p<0.0001). We found that at 5 dpi, rhgal9 treated mice exhibited enhanced viral clearance in the BAL but not in the lung parenchyma. Additionally, we found increased CD8 T cell (p<0.001) and decreased neutrophil (p<0.05) frequencies in the lung at 5 dpi. Finally, we found that BAL fluid had elevated levels of Type 1 Interferon [IFNa (p<0.01) and IFNb (p<0.01)] at 2 dpi and increased MyD88 proinflammatory cytokines [IL1a (p<0.05), IL1b (p<0.01), TNFa (p<0.05), and MIP1a (p<0.05) at 5 dpi. Conclusion: Our study suggests that rhgal9 treatment may be potentially therapeutic for treating acute COVID19. Our data suggest that rhgal9 treatment in combination with other anti-inflammatory mediators may curtail damaging inflammation associated with SARS-CoV2 disease. Further studies are required to determine the optimal time, combination and duration of treatment pi to effectively target the gal9 pathways.

Umbilical cord-derived mesenchymal stem cell transplantation for COVID -19 patients: long-term benefits for lung regeneration

Coronavirus disease 2019 (COVID-19) has affected hundreds of millions of people worldwide. Currently, mortality due to COVID-19 is significantly reduced by vaccination, antiviral drugs, and some improved treatments. Mesenchymal stem cell (MSC) transplantation—particularly umbilical cord-derived MSC (UC-MSC)—has been used as an adjuvant therapy for COVID-19 with some clinical evidence (reviewed in the publication). Moreover, a recent piece published in eBiomedicine (part of The Lancet, https://doi.org/10.1016/j.ebiom.2021.103789) in the previous month showed the long-term effects of UC-MSC transplantation in COVID-19 in a 1-year follow-up randomized, double-blind, placebo-controlled trial, demonstrating significantly recovered lung lesions and symptoms compared to the control group (i.e., without UC-MSC transplantation). In this commentary, we would like to discuss the value of UC-MSC transplantation for COVID-19 patients based on the results from this study and suggest applying this therapy for COVID-19 patients.

Cancer patients display diminished viral RNA clearance and altered T cell responses during SARS-CoV-2 infection

Cancer patients display immunomodulation related to malignancy and anti-cancer therapies, but how these factors impact COVID-19 remains unknown. To investigate immune responses in cancer patients with COVID-19, we undertook a prospective case-control study, enrolling hospitalized solid tumor patients with acute COVID-19, as well as age-, gender-, and comorbidity-matched COVID-19 patients without cancer as controls. Using biospecimens collected during hospitalization, we performed virologic measurements as well as in-depth immunophenotyping of cellular, antibody and cytokine responses. We enrolled 17 cancer patients (cases) admitted to Yale-New Haven Hospital between March 15 and June 30, 2020 with COVID-19, as well as 17 matched non-cancer patients (controls) admitted with COVID-19. No significant differences were observed between cases and controls based on patient characteristics (age, gender, race, co-morbidities, smoking history, days from symptom onset to COVID-19 diagnosis) or outcomes (COVID-19 severity, length of hospital stay, rate of intubation or mortality). The most common primary tumor sites were lung (4/17) and gastrointestinal (4/17);all cases had received cancer-directed therapy within 6 months of COVID-19 diagnosis, with 13/17 receiving treatment less than 1 month prior to hospitalization. Three of 17 cases had received immune checkpoint inhibitor therapies. Despite having similar SARS-CoV-2 viral RNA loads at the time of COVID-19 diagnosis when compared with controls, cancer cases had increased viral RNA abundance during hospitalization, suggesting slower clearance. Antibody responses against SARS-CoV-2 were preserved in cancer cases, with cases displaying similar levels of IgM and IgG antibodies directed against SARS-CoV-2 epitopes compared to controls. Cytokine profiling revealed higher plasma levels of CCL3, IL1A and CXCL12 in cancer cases compared to controls. Using flow cytometric immunophenotyping, we found that innate immune and non-T cell adaptive immune parameters were similar between cases and controls hospitalized with COVID-19. However, among cancer cases on conventional therapies, T cell lymphopenia was more profound, and these cases demonstrated higher levels of CD8+ exhausted (CD8+CD45RA-PD1+TIM3+ ), CD8+GranzymeB+ and CD4+CD38+HLA-DR+ and CD8+CD38+HLA-DR+ activated T cells when compared with controls;interestingly, these differences were not observed in patients who had received immune checkpoint inhibition. Thus, we found reduced viral RNA clearance and specific alterations in T cell and cytokine responses in cancer patients hospitalized with COVID-19 compared with matched controls with COVID-19. This dysregulated T cell response in cancer patients, which may reflect immune modulation due to chronic antigen stimulation as well as cancer therapies, may lead to altered virologic and clinical outcomes in this population.

Mucosal Cytokine Profiles in Children with COVID-19

Background. The mechanisms associated with COVID-19 in children are not well understood. We sought to define the differences in nasopharyngeal (NP) cytokine profiles according to clinical presentation in children with COVID-19. Methods. Single-center, prospective study in 137 children and adolescents < 21 years of age hospitalized with COVID-19, and 35 age, sex and race matched pre-pandemic (2016-2019) healthy controls. Children with COVID-19 were categorized according to their clinical presentation in: COVID-19-symptomatic;COVID-19-screening, and multisystem inflammatory syndrome (MIS-C). NP swabs were obtained within 24 hours of admission to measure SARS-CoV-2 loads by rt-PCR, and a 92-cytokine panel. Unsupervised and supervised analysis adjusted for multiple comparisons were performed. Results. From 3/2020 to 1/2021, we enrolled 76 COVID-19-symptomatic children (3.5 [0.2-15.75] years);45 COVID-19-screening (11.1 [4.2-16.1] years), and 16 MIS-C (11.2 [5.9-14.6] years). Median NP SARS-CoV-2 loads were higher in COVID-19-symptomatic versus screening and MIS-C (6.8 vs 3.5 vs 2.82 log10 copies/mL;p< 0.001). Statistical group comparisons identified 15 cytokines that consistently differed between groups and were clustered in three functional categories: (1) antiviral/regulatory, (2) pro-inflammatory/chemotactic, and (3) a combination of (1) and (2);(Fig 1). All 15 cytokines were higher in COVID-19-symptomatic versus controls (p< 0.05). Similarly, and except for TNF, CCL3, CCL4 and CCL23, which were comparable in COVID-19-symptomatic and screening patients, the remaining cytokines were higher in symptomatic children (p< 0.05). PDL-1 (p=0.01) and CCL3 (p=0.03) were the only cytokines significantly decreased in children with MIS-C versus symptomatic COVID-19 children. The 15 cytokines identified by multiple comparisons were correlated using Person's in R software. Red reflects a positive correlation and blue a negative correlation with the intensity of the color indicating the strength of the association. Conclusion. Children with symptomatic COVID-19 demonstrated higher viral loads and greater mucosal cytokines concentrations than those identified via screening, whereas in MIS-C concentrations of regulatory cytokines were decreased. Simultaneous evaluation of viral loads and mucosal immune responses using non-invasive sampling could aid with the stratification of children and adolescents with COVID-19 in the clinical setting.

Longitudinal Plasma Cytokine Profiles Differentiating COVID-19 Severity Groups

Background. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), an infection with widely varying clinical severity. Severe COVID-19 was initially proposed to be secondary to cytokine storm syndrome (CSS). However, studies since showed that patients with severe COVID-19 rarely display CSS cytokine phenotypes, and may have more limited inflammatory responses instead. Methods. Prospective cohorts, aged 0-90 years of age who tested positive by polymerase chain reaction (PCR) for SARS-CoV-2 were enrolled from inpatient hospitals and outpatient testing centers in Memphis, TN from May 2020-January 2021. Longitudinal blood samples were obtained including acute, sub-acute and convalescent timepoints. Severity scores of asymptomatic, mild, moderate, and severe COVID-19 were assigned at time of convalescent assessment. Plasma was analyzed with a quantitative human magnetic 38-plex cytokine assay. Results. : 169 participants were enrolled, including 8 asymptomatic, 117 mild, 22 moderate and 17 severe cases, and 5 children with post-COVID-19 multisystem inflammatory syndrome in children (MIS-C). All moderate and severe patients were hospitalized and received treatment (39%). Clear distinctions were seen between asymptomatic-mild cases and moderate-severe cases at acute timepoints and during disease progression for GCSF, IL-8, IL-10, IL-15, IL-1Ra, IP-10, MIP-1a, MIP-1β, and TGFα. There was a significant difference between participants who did and did not require hospitalization for acute timepoint levels of IL-10, IL-15, MIP-1 β and TGFα (p< 0.01). Only 4 participants with active COVID-19 were found to meet criteria for CSS (2%), only 3 of which were severe. MIS-C participants showed nearly universally elevated cytokine levels compared to those with active COVID-19. Conclusion. Moderate and severe acute COVID-19 has a distinct cytokine profile from asymptomatic and mild cases, as detected from acute, subacute and convalescent plasma.

Epidemiology, virology, clinical features, diagnosis, and treatment of SARS-CoV-2 infection

Since December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged and spread quickly worldwide. The disease is generally mild in adult people but in any with comorbidities may proceed to acute respiratory distress syndrome (ARDS), pneumonia, and multi-organ dysfunction. By performing molecular tests on respiratory secretions can diagnose the virus. Elevated C-reactive protein (CRP) and normal/low white cell counts are common laboratory diagnoses of COVID-19 while the tomographic chest scan is usually irregular for many infected people. Some patients progress to respiratory failure, pneumonia, and finally death by the end of the first week of illness because of the sharp rise in inflammatory cytokines such as IL7, IL2, GCSF, IL10, MIP1A, MCP1, IP10, and TNFα. Various approaches to the COVID- 19 are being performed by scientists. Use of chemical medical drugs that are effective for other viral infections. Among them, remdesivir was approved by FDA on 1th May 2020 because of its impact to treat patients. Also, several studies have revealed that many Chinese herbal remedies have a remarkable impact on the healing process when simultaneously were used along with pharmacological drugs. In the meantime, many efforts have been made to produce an effective vaccine, and so far, the Ad5-vectored COVID-19 vaccine has been successful and has entered phase 2 in the human trial. The current review focus on epidemiology, virology, clinical features, diagnosis, and available treatment of coronavirus that might assist researchers and clinicians in establishing action options for timely against this infection.

ORAL TANNINS REDUCE PROINFLAMMATORY CYTOKINES ASSOCIATED WITH DIARRHEA AND PNEUMONIA IN HOSPITALIZED COVID-19 PATIENTS

INTRODUCTION: There is evidence that the gut microbiota and its relationship with the immune system could be involved in the pathogenesis of COVID-19. SARS-CoV-2 can cause gastrointestinal symptoms during the early phases of the disease. Intestinal dysfunction induces changes in intestinal microbes, and an increase in inflammatory cytokines. Therefore, microbiota modulation could play a role in COVID-19 treatment. Tannins have been shown to work as prebiotics on the gastrointestinal microbiota. In particular, quebracho and chestnut tannins have shown to regulate the immune response and decrease in vitro-cytokines production, through microbiota fermentation-secondary metabolites, such as quercetin and SCFAs. OBJECTIVE: To evaluate the efficacy and the effect on cytokine levels of a tannin specific natural extract in COVID-19 patients. MATERIAL AND METHODS: This prospective, doubleblind, and randomized study was approved by the Hospital de Clínicas, José de San Martín (Buenos Aires, Argentina). Blood and stool samples were collected at baseline (Day 0) and after treatment (Day 14) during July-October 2020, with final follow-up in November 2020. We randomly assigned 124 RT-PCR confirmed COVID-19 cases (>18 years) to receive oral dry extracts of quebracho and chestnut tannins (240 mg) and B12 vitamin (0.72 μg) or placebo, twice daily for 14 days as adjunct treatment to their standard of care management. 27-pro and anti-inflammatory cytokines were measured on day 0 and 14 (Bio-Plex Pro™, Bio-Rad). Final enrollment of 140 patients with matched fecal microbiome characterization (16S, WGS and metabolites) is expected. RESULTS. Of 124 patients who were randomized (mean age 55+/-15, 63 [50.81%] male), 121 (97.58%) completed the trial. No adverse events were observed in the tannin group. Patients presenting with diarrhea (13%) had a trend to have elevated blood MIP-1α levels, which were significantly reduced by tannin treatment (Table 1). At baseline, higher levels of MIP-1α were also associated with diagnosis of pneumonia (Fig. 1), which was maintained after adjusting for confounders (age, sex, diabetes;p=0.04). Moreover, at baseline there was a positive correlation between MIP-1 α and IL-1ra, IL-2, MIP-1b and TNF-α, with all of these cytokines decreasing mostly with tannin treatment. CONCLUSION: To our knowledge, this clinical trial represents the first study to target the gut microbiome in hospitalized COVID-19 patients. Oral tannins as adjunct treatment with standard-of-care management of these patients significantly reduced proinflammatory cytokine levels that are generally associated with poor predictive outcomes, i.e. pneumonia and diarrhea. Further, our prospective studies will determine which microbiome-mediated mechanisms may attenuate the cytokine storm that is evident in COVID-19 disease pathogenesis. (Table presented) (Figure presented)