Pre-existing chronic liver disease is associated with poor outcome in patients with SARS-CoV2 infection

COVID-19 is characterized by predominant respiratory and gastrointestinal symptoms. Liver enzymes derangement is seen in 15-55% of the patients. Cirrhosis is characterized by immune dysregulation, leading to concerns that these patients may be at increased risk of complications following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Patients with metabolic dysfunction-associated fatty liver (MAFLD) had shown a 4-sixfold increase in severity of COVID-19, and its severity and mortality increased in patients with higher fibrosis scores. Patients with chronic liver disease had shown that cirrhosis is an independent predictor of severity of COVID-19 with increased hospitalization and mortality. An international European registry study included 756 patients with chronic liver disease from 29 countries reports high mortality in patients with cirrhosis (32%). Data of 228 patients collected from 13 Asian countries on patients with CLD, known or newly diagnosed, with confirmed COVID-19 (APCOLIS study) showed that SARSCoV- 2 infection produces acute liver injury in 43% of CLD patients without cirrhosis. Additionally, 20% of compensated cirrhosis patients develop either ACLF or acute decompensation. In decompensated cirrhotics, the liver injury was progressive in 57% of patients, with 43% mortality. Patients with CLD and associated diabetes and obesity had a worse outcome. Liver related complications were seen in nearly half of the decompensated cirrhotics, which were of greater severity and with higher mortality. Increase in Child Turcotte Pugh (CTP) score and model for end-stage liver disease (MELD) score increases the mortality in these patients. In a subsequent study of 532 patients from 17 Asian countries was obtained with 121 cases of cirrhosis. An APCOLIS risk score was developed, which included presence of comorbidity, low platelet count, AKI, HE and respiratory failure predicts poor outcome and an APCOLIS score of 34 gave a sensitivity and specificity of 79.3%, PPV of 54.8% and NPV of 92.4% and predicted higher mortality (54.8% vs 7.6%, OR = 14.3 [95 CI 5.3-41.2], p<0.001) in cirrhosis patients with Covid-19. The APCOLIS score is helpful in triaging and prognostication of cirrhotics with Coivd-19. The impact of COVID-19 on patients with cirrhosis due to non-alcoholic fatty liver disease (NASH-CLD) was separately studied in 177 NASH-CLD patients. Obese patients with diabetes and hypertension had a higher prevalence of symptomatic COVID. Presence of diabetes [HR 2.27], fraility [HR 2.68], leucocyte counts [HR 1.69] and COVID-19 were independent predictors of worsening liver functions in patients with NASH-CLD. Severity of Covid in Cirrhosis could also be assessed by measuring ICAM1 the Intercellular Adhesion Molecule, an indicator of Endothelial Injury Marker. in Cirrhosis with Covid 19 Immunosuppression should be reduced prophylactically in patients with autoimmune liver disease and post-transplantation with no COVID-19. Hydroxychloroquine and remdesivir are found to be safe in limited studies in a patient with cirrhosis and COVID-19. And is safe in cirrhosis patients. However, flare of AIH has been reported in AIH patients. For hepatologists, cirrhosis with COVID-19 is a pertinent issue as the present pandemic cause severe disease in patients with chronic liver disease leading to more hospitalization and decompensation.

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).

ICAM-1 MEDIATES PLASMACYTOID DENDRITIC CELL SENSING OF SARS-CoV-2-INFECTED CELLS

Background: Plasmacytoid dendritic cells (pDCs) are the major producer of type I IFNs (IFN-I), the critically important antiviral cytokines against SARS-CoV- 2. Although pDCs can sense cell-free SARS-CoV-2 virions, it is unknown whether they can detect infected cells to produce IFN-I. Since cell-to-cell transmission accounts for 90% of SARS-CoV-2 infections (Zeng et al., 2022), we examined the relevance of pDC sensing of infected cells in SARS-CoV-2 infection and whether the virus exploits this pathway to evade IFN-I responses. Method(s): LSPQ1, the first SARS-CoV-2 clinical isolate received from the Public Health Laboratory of Quebec, was used as a prototype virus. SARS-CoV-2 variants of concerns (VOCs) were also used. PBMCs or enriched pDCs were cocultured with mock-infected or SARS-CoV-2-infected HeLa-hACE2 or Calu-3. Either PBMCs, enriched pDCs, or HeLa-hACE2 were pretreated with anti-human ICAM-1 antibody or isotype control. The conjugate formation was determined by flow cytometry. Polarized Caco cells were used to validate critical data. Result(s): Upon sensing infected cells, PBMCs release 6-fold more IFN-I than they do when exposed to cell-free virions. Antibody-mediated depletion of pDCs from PBMCs abolishes IFN-I secretion. Direct contact of pDCs with infected cells is required for sensing since the use of a transwell membrane reduces IFN-I release by 85%. Infected cells form conjugates with pDCs more frequently (3.2-fold higher) than uninfected cells. Blocking ICAM-1 on infected cells or pDCs impacts conjugate formation and significantly suppresses IFN-I production by 55-80%, suggesting bidirectional interaction. Moreover, human lung cells infected with VOCs are sensed to a different extent with the alpha variant being the least efficiently sensed by pDCs compared to the delta or omicron strains. Even though SARS-CoV-2 is primarily released from the apical domain of polarized infected Caco cells, sensing of infected cells does occur upon direct contact of pDCs with the basolateral domain, highlighting how pDCs antiviral responses might be triggered in respiratory tissues. Conclusion(s): pDC sensing of infected cells accounts for the vast majority of IFN-I released during SARS-CoV-2 infection. ICAM-1 promotes physical contact between pDCs and infected cells, thus leading to efficient sensing. Differential pDC sensing of SARS-CoV-2 VOC-infected cells suggests that some VOCs might manipulate the interactions of pDCs with infected cells to limit IFN-I responses.

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.

A review of fibroblast-like synoviocytes in the pathogenesis of Rheumatoid arthritis: Their activation and the inhibition of their apoptosis

Rheumatoid Arthritis (RA) is a systemic, autoimmune, inflammatory disease characterized by synovial hyperplasia, inflammatory cell infiltration in the synovial tissues, and progressive destruction of cartilage and bones. This disease often leads to chronic disability. More recently, activation of synovial fibroblasts (SFs) has been linked to innate immune responses and several cellular signalingpathways that ultimately result in the aggressive and invasive stages of RA. SFs are the major sources of pro-inflammatory cytokines in RA synovium. They participate in maintaining the inflammatory state that leads to synovial hyperplasia and angiogenesis in the inflamed synovium. The altered apoptotic response of synovial and inflammatory cells has been connected to these alterations of inflamed synovium. RA synovial fibroblasts (RASFs) have the ability to inhibit several apoptotic proteins that cause their abnormal proliferation. This proliferation leads to synovial hyperplasia. Apoptotic pathway proteins have thus been identified as possible targets for modifying the pathophysiology of RA. This review summarizes current knowledge of SF activation and its roles in the inhibition of apoptosis in the synovium, which is involved in joint damage during the effector phase of RA development.Copyright © 2022 Biomedpress.

Jing Si Herbal Drink as a prospective adjunctive therapy for COVID-19 treatment: Molecular evidence and mechanisms

Background: SARS-CoV-2 has led to a sharp increase in the number of hospitalizations and deaths from pneumonia and multiorgan disease worldwide;therefore, SARS-CoV-2 has become a global health problem. Supportive therapies remain the mainstay treatments against COVID-19, such as oxygen inhalation, antiviral drugs, and antibiotics. Traditional Chinese medicine (TCM) has been shown clinically to relieve the symptoms of COVID-19 infection, and TCMs can affect the pathogenesis of SARS-CoV-2 infection in vitro. Jing Si Herbal Drink (JSHD), an eight herb formula jointly developed by Tzu Chi University and Tzu Chi Hospital, has shown potential as an adjuvant treatment for COVID-19 infection. A randomized controlled trial (RCT) of JSHD as an adjuvant treatment in patients with COVID-19 infection is underway Objectives: This article aims to explore the efficacy of the herbs in JSHD against COVID-19 infection from a mechanistic standpoint and provide a reference for the rational utilization of JSHD in the treatment of COVID-19. Method(s): We compiled evidence of the herbs in JSHD to treat COVID-19 in vivo and in vitro. Result(s): We described the efficacy and mechanism of action of the active ingredients in JSHD to treat COVID-19 based on experimental evidence. JSHD includes 5 antiviral herbs, 7 antioxidant herbs, and 7 anti-inflammatory herbs. In addition, 2 herbs inhibit the overactive immune system, 1 herb reduces cell apoptosis, and 1 herb possesses antithrombotic ability. Conclusion(s): Although experimental data have confirmed that the ingredients in JSHD are effective against COVID-19, more rigorously designed studies are required to confirm the efficacy and safety of JSHD as a COVID-19 treatment.Copyright © 2021

Endothelial Biomarkers in Critically Ill Patients with Covid-19: Potential Predictors of the Need for Dialysis

Introduction: Endothelial dysfunction and coagulopathy have been reported as the basis of severe Acute Kidney Injury (AKI) associated with COVID-19. Endothelial biomarkers can detect kidney damage early and allow the adoption of efficient measures to prevent the progression of the disease and its complications. This finding could facilitate the follow-up of patients at higher risk, as well as provide early diagnosis strategies and promote the rational use of resources. The aim of this study is to assess the role of vascular biomarkers to predict the need for hemodialysis in critically ill patients with COVID-19. Method(s): This is a prospective study with 58 patients critically ill due to COVID-19 infection admitted to a tertiary hospital in Fortaleza, Northeast Brazil, from 2020 to 2021. General laboratory tests and vascular biomarkers such as VCAM-1, Syndecan-1, ACE-2, ICAM-1, Angiopoietin-1 and Angiopoietin-2 were quantified on admission to the intensive care unit (ICU). Result(s): There was a 40% mortality rate. VCAM and the Ang-2/Ang-1 ratio at ICU admission were associated with the need for hemodialysis. Vascular biomarkers (VCAM-1, Syndecan-1, angiopoietin-2/anogiopoietin-1 ratio) and thrombocytopenia were predictors of dialysis, and their cutoff values were useful to stratify patients with worse prognosis in the Kaplan-Meier analysis. In the cox multivariate regression analysis with models adjusted according to the presence or absence of platelets, VCAM-1 [O.R. 1.13 (95% CI: 1.01 - 1.27);p=0.034] was an independent predictor of dialysis in all models, and the Ang-2/Ang-1 ratio [O.R. 4.87 (95% C.I.: 1.732 - 13.719);p=0.003] was associated with the need for dialysis in the model without platelet input. Conclusion(s): Vascular biomarkers, mainly VCAM-1 and Ang-2/Ang-1 ratio, and coagulation disorders showed important predictive value for the need for hemodialysis in critically ill patients with COVID-19. No conflict of interestCopyright © 2023

ACE2 IS INVOLVED IN SARS-COV-2 INDUCED ENDOTHELIAL CELL INFLAMMATION INDEPENDENT OF VIRAL REPLICATION

Objective: COVID-19 association with cardiovascular disease is thought to be due to endothelial cell inflammation. ACE2 interactions with SARS-CoV-2 spike protein S1 subunit is important to viral infection. Here we questioned whether SARS-CoV-2 induces vascular inflammation via ACE2 and whether this is related to viral infection. Design and Methods: Human microvascular endothelial cells (EC) were exposed to recombinant S1p (rS1p) 0.66 ug/mL for 10 min, 5 h and 24 h. Gene expression was assessed by RT-PCR and levels of IL6 and MCP1, as well as ACE2 activity, were assessed by ELISA. Expression of ICAM1 and PAI1 was assessed by immunoblotting. ACE2 activity was blocked by MLN4760 (ACE2 inhibitor) and siRNA. Viral infection was assessed by exposing Vero E6 (kidney epithelial cells;pos ctl) and EC to 105 pfu of SARS-CoV-2 where virus titre was measured by plaque assay. Results: rS1p increased IL6 mRNA (14.2 ± 2.1 vs. C:0.61 ± 0.03 2-ddCT) and levels (1221.2 ± 18.3 vs. C:22.77 ± 3.2 pg/mL);MCP1 mRNA (5.55 ± 0.62 vs. C:0.65 ± 0.04 2-ddCT) and levels (1110 ± 13.33 vs. C:876.9 ± 33.4 pg/mL);ICAM1 (17.7 ± 3.1 vs. C:3.9 ± 0.4 AU) and PAI1 (5.6 ± 0.7 vs. C: 2.9 ± 0.2), p < 0.05. MLN4760, but not rS1p, decreased ACE2 activity (367.4 ± 18 vs. C: 1011 ± 268 RFU, p < 0.05) and blocked rS1p effects on ICAM1 and PAI1. ACE2 siRNA blocked rS1p-induced IL6 release, ICAM1, and PAI1 responses as well as rS1p-induced NFkB activation. EC were not susceptible to SARS-CoV-2 infection, while the virus replicated well in Vero E6. Conclusion: rS1p induces an inflammatory response through ACE2 in endothelial cells;an effect that was independent of viral infection.

THE ROLE of IL-6 in ENDOTHELIAL DYSFUNCTION: RHEUMATOID ARTHRITIS and COVID-19, TWO PATHOGENIC MODELS in COMPARISON

Background: Rheumatoid arthritis (RA) is the most common systemic autoimmune disease that primarily affects joints but is also often characterized by extra-articular involvement1. Cardiovascular diseases are the most important causes of sudden death in these patients, which present a risk of developing cardiovascular events increased by 48%2. The causes of increased cardiovascular risk are several and not completely understood, but recent evidence supports the key role of endothelial dysfunction in pathogenesis. In this complex scenario, it is known that IL-6 receptors are present at the endothelial level and can be activated leading to endothelial dysfunction. SARS-Cov-2 is a coronavirus responsible for the disease called 'coronavirus disease 2019' (CoViD-19) characterized by clinical manifestations ranging from a flu-like syndrome up to severe lung damage associated with systemic hyper cytokine syndrome that can lead to multiple organ failure and death. Therefore, both RA and Covid-19 are associated with an increased pro-thrombotic and cardiovascular risk and IL-6 might be crucial in the patho-physiological mechanisms of both diseases. Objectives: The main hypothesis of this study was to evaluate the possible role of IL-6 as a promoter of endothelial dysfunction in RA and CoViD-19. Methods: In vitro experiments were conducted on the endothelial cell line EA. hy926. Cells were treated for 24 h with fetal bovine serum (FBS), a pool of RA patients' sera or a pool of CoViD-19 patients' sera. The expression levels of adhesion molecules (V-CAM1/CD-106, I-CAM/CD-54, p-selectine/CD-62, tissue factor/CD-142) and apoptosis were analyzed using cytofuorimetric technique. In addition, the autophagy level, using the autophagy markers p62 and LC3II, were evaluated through a western-blot analysis. The same experiments were conducted co-treating cells with the same pool of sera in addition to tocilizumab (TCZ), an anti-IL-6 drug, to verify the reversibility of the process and test the role of the aforementioned cytokine. Data are reported as interquartile median values. The Kruskal Wallis test was used for unpaired samples and the Mann-Whitney test for paired samples. P<0.05 values were considered statistically signifcant. Results: EA. hy926 cells, when treated with both RA and CoViD-19 patients' sera, showed increased levels of activation molecules and apoptosis compared to FBS treated cells. In addition, we observed increased levels of both p62 and LC3 proteins after both rheumatoid arthritis and CoViD-19 patients' sera treatment. All these fndings were reversible in the presence of TCZ. The results are presented in Figure 1. Conclusion: Our data showed that treatment with RA and CoViD-19 patients' sera increase the activation and death of endothelial cells in vitro. The increased level of cells death is possibly due to a block of autophagy. The reversibility of the process after blocking IL-6 with TCZ co-treatment confrms the hypothesis that IL-6 can play a key role in the pathogenesis of endothelial damage in patients with RA and CoViD-19.

ENDOTHELIAL CELLS AND BLOOD VESSELS – CRITICAL TARGETS FOR COVID-19-INDUCED TISSUE INJURY AND SPREAD TO DISTANT ORGANS. UNDERLYING MECHANISMS INCLUDE ENDOTHELIAL DYSFUNCTION AND INJURY

Endothelial cells (ECs) lining the blood vessels of all organs express the SARS-CoV2 receptor. In the absence of preexisting tissue damage, the virus would need to pass through the ECs to blood vessels to infect other tissues. Thus, EC are a target for SARS-CoV-2 infection and a conduit for viral dissemination to distant organs. We hypothesized that ECs infection and/ or injury are the mechanisms of COVID-19 pathology and multi-organ dissemination and injury. Methods: Human studies: We used lung, heart, kidney, and small bowel specimens obtained during autopsies (n=5) from COVID-19 patients and uninfected subjects. Studies: 1) histologic evaluation of endothelial damage and endotheliitis, 2) immunohistochemistry for vWF, PAI-1, VCAM-1, & ICAM-1. Studies in cultured human microvascular ECs (HMVECs): We cultured lung and cardiac HMVECs in the presence or absence of SARSCoV- 2 S1 and/or S2 protein (10 ng/ml) for 0 - 24 hr. Studies:1) cell viability and proliferation;2) angiogenesis on Matrigel and cell migration;3) mitochondrial membrane potential (MMP);4) RNA seq analysis;5) Western blotting for vWF, PAI-1, VCAM-1, and ICAM-1. We examined the protective effect of melatonin, Coenzyme Q10 and nerve growth factor on S1/S2 protein induced HMVEC cell damage. Results: Histopathologic examination revealed presence of endothelial abnormalities and endotheliitis with marked presence of inflammatory cells in vessel wall & lumen, and fibrinous microthrombi) in lung, heart & kidney in autopsy specimens of COVID-19 patients. Immunostaining visualized increased vWF, PAI-1, VCAM- 1, & ICAM-1 in COVID-19. In in vitro study, S1 and S2 proteins induced endothelial injury, reduced angiogenesis and phosphorylated/activated Erk and Akt proteins in cultured HMVECs. Treatment of HMVECs for 1 & 4 hours with S2 but not S1 protein increased ICAM-1 levels by 1.4- to 1.8-fold (P < 0.001). RNA Seq analysis showed that treatment of HMVECs with S1 and S2 proteins upregulated VCAM-1, ICAM-1 and E-selectin mRNA in cultured HMVECs. Melatonin, Coenzyme Q10 and NGF stimulated angiogenesis in HMVECs by 2.4-, 1.3-&1.4-fold (all P < 0.001). Conclusions: 1) Significant endothelial abnormalities, blood vessel damage and endotheliitis are present in lung, heart and kidney autopsy specimens of COVID-19 patients, 2) There is increased expression of vWF, PAI-1, VCAM-1, and ICAM- 1 in lung, heart, and kidney specimens of COVID-19 patients, 3) Treatment of cultured HMVECs with SARS-CoV-2 S1 and S2 proteins upregulates VCAM-1, ICAM-1 and Eselectin expression, 4) SARS-CoV-2 S1 and S2 proteins induce endothelial injury in cultured HMVECs, and 5) melatonin, Coenzyme Q10 and NGF stimulated EC function. These studies uncovered novel mechanism – endothelial dysfunction underlying SARS-CoV-2 and identified melatonin, Coenzyme Q10 and NGF as potential drugs for treatment of COVID- 19-induced EC injury

A Novel Ogremorphin Group of First-in-Class GPR68 Inhibitors Upregulate Endothelial Barrier Function and Rescue Bacterial Pathogens or Acidosis-Induced Inflammation in Lung Endothelium

Rationale: An increase in endothelial permeability resulting from the disruption of endothelial barrier and aggravated inflammatory responses are two major pathological hallmarks of various lung disorders including the current global pandemic COVID-19. Drugs that enable the preservation and restoration of endothelial function represent attractive therapeutic targets to treat endothelial dysfunction-derived cardiopulmonary diseases. A role of G protein-coupled receptors (GPCRs), especially a sub-family of proton-sensing GPCRs including GPR4 and GPR68, has been suggested in modulation of endothelial function. In this study, we analyzed the barrier protective and anti-inflammatory effects of two recently developed novel class of GPR68 inhibitors: ogremorphins OGM8345 and OGM-1.Methods: Transendothelial electrical resistance (TER) was monitored in human pulmonary arterial endothelial cells (HPAECs) to evaluate endothelial barrier function. Quantitative real time PCR and western blot analyses were performed to determine mRNA and protein expression of endothelial inflammation markers, respectively. Acidic pH (6.5) medium was used to induce acidosis, and luciferase-based Tango assay was employed to evaluate GPR68 activation. C57BL/6 mice were exposed to lipopolysaccharide (LPS from Escherichia coli) or heatkilled Staphylococcus aureus (HKSA), and vascular leak/inflammation was assessed by determining the extravasation of intravenously injected Evans blue tracer into lungs and total cells/protein count in bronchoalveolar lavage samples. Results: A robust dose-dependent increase in basal EC barrier function was observed with OGM8345 (1-5 μM) and OGM-1 (0.3-1.5 μM) evident by an 150-200% increase in TER values. Both inhibitors also effectively rescued LPS- and HKSA-induced EC hyperpermeability. RT-PCR analysis demonstrated that LPS or HKSA-induced upregulation of inflammatory cytokines/chemokines genes TNF-α, ICAM-1, VCAM-1, IL-6, IL-8, IL- 1β, and CXCL5 was significantly attenuated by OGMs. Consistently, both OGMs suppressed LPSand HKSA-induced protein expression of VCAM-1 and ICAM-1. In contrast, pharmacologic inhibition of GPR4 by NE 52-QQ57 failed to alleviate LPS or HKSA-induced EC barrier dysfunction and inflammation. Importantly, LPS, HKSA or acidosis stimulation resulted in increased GPR68 mRNA expression and GPR68 activity that was inhibited by OGMs. Intratracheal injection of LPS or HKSA in C57BL/6 mice caused vascular leak and lung inflammation that was attenuated by both OGMs as illustrated by reduced Evans blue accumulation in the lungs and significant inhibition of accumulation of inflammatory cells and protein content in bronchoalveolar lavage samples. Conclusion: These results establish a critical role of GPR68 in endothelial dysfunction and strongly suggest a therapeutic potential of GPR68-selective inhibitors in improving endothelial dysfunction caused by bacterial infections and acidosis associated with acute and chronic lung injury.

Genetic and structure of novel coronavirus COVID-19 and molecular mechanisms in the pathogenicity of coronaviruses

The recently identified 2019 novel coronaviruses (2019-nCoV) has caused extra-human infections. 2019-nCoV identified a global threat that is causing an outbreak of unusual viral pneumonia in patients with severe acute respiratory syndrome (SARS)-coronaviruses 2 (SARS-CoV-2). Considering the relatively high identity of the receptor-binding domain (RBD) in 2019-nCoV and SARS-CoV, it is urgent to assess the cross-reactivity of anti-SARS-CoV antibodies with 2019-nCoV spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against 2019-nCoV. The zinc metallopeptidase angiotensin-converting enzyme 2 (ACE2) is the only known human homolog of the key regulator of blood pressure ACE. ACE2 also serves as the cellular entry point for the SARS virus, therefore, a prime target for pharmacological intervention. SARS-CoV-2 uses the SARS-CoV receptor for entry and the serine protease transmembrane protease serine 2 for spike (S) protein priming. That it is still necessary to develop novel mAbs that could bind specifically to 2019-nCoV RBD. Cell entry of coronaviruses depends on the binding of the viral S proteins to cellular receptors and S protein priming by host cell proteases. A transmembrane protease serine 2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention. This review will help understand the biology and potential risk of CoVs that exist in richness in wildlife such as bats. We provide a brief introduction to the pathogenesis of SARS-CoV and Middle East respiratory syndrome-CoV and interaction between the RBD of coronavirus spike protein and ACE2.

Profiling viral receptors in nasal and amniotic samples at birth

Introduction/Aim: Children with wheeze and asthma present with airway epithelial vulnerabilities, such as impaired responses to viral infection. It is postulated that the in utero environment may contribute to the development of such airway epithelial vulnerabilities, that may predispose children to wheeze and asthma outcomes. To explore developmental mechanisms, further research is required using epithelial samples at birth. Our study asked whether amniotic epithelial samples from placentas show similar viral receptor expression to nasal epithelial cells at birth. We aimed to investigate expression of respiratory viral receptors for human rhinovirus (HRV), respiratory syncytial virus (RSV) and COVID-19-causing coronavirus (SARS-CoV-2) in nasal and amniotic epithelial samples. Methods: Unmatched nasal (n = 20 births) and amniotic (n = 33 newborns) epithelial samples were collected from ORIGINS cohort participants recruited into the AERIAL study. Using purified RNA, receptor expression for HRV (ICAM-1, LDLR, CDHR3), RSV (NCL, TLR4) and SARSCoV- 2 (ACE2, TMPRSS2) was assessed by qPCR. In addition, receptor protein expression was quantified through western blot and localized using immunohistochemistry in amniotic samples only. Results: Nasal epithelial and amniotic samples expressed various receptors for HRV, RSV and SARS-CoV-2 at the gene level in nasal (median(IQR) arbitrary units (AU);ICAM-1: 11.44(63.18);LDLR: 4.00(7.32);CDHR3: 0.40 (1.14);NCL: 2.32(2.18);CX3CR1: 2.17(2.33);TLR4: 2.20 (6.20);TMPRSS2: 1.99(4.85);ACE2: 0.36(0.52) AU) and amnion (ICAM-1: 0.69(2.21);LDLR: 0.39(1.38);CDHR3: 1.0 x 10-4(3.0x10-4);NCL: 1.03(0.55);CX3CR1: 0.12(0.24);TLR4: 0.10(0.13);TMPRSS2: 3.0 x 10-4 (16.0x10-4);ACE2: 0.01(0.02) AU). Amniotic samples also expressed these receptors at the protein level (ICAM-1: 0.03(0.05);LDLR: 0.06(0.03);CDHR3: 0.28(0.15);NCL: 0.96(1.19);CX3CR1: 0.08(0.08);TMPRSS2: 0.09(0.06);ACE2: 0.34(0.92) AU) and expression within the amniotic epithelium was confirmed by immunohistochemistry. Conclusion: Newborn nasal and amniotic epithelial samples expressed receptors for respiratory viruses, HRV, RSV, SARS-CoV-2. These findings warrant further investigation of the clinical significance of receptor expression in relation to prenatal and postnatal exposures, as well as childhood asthma development.

Host Protein Biomarkers Predicting Severity of Lung Damage due to COVID-19

Background. Biomarkers to predict the severity of lung damage due to COVID-19 are urgently needed to inform management and treatment decisions. Our objective was to investigate the predictive value of host proteins for worsening respiratory failure in one of the by COVID-19 most affected and diverse patient populations in the US. Methods. We performed a prospective single-center cross-sectional study of 34 adult patients admitted to Montefiore Medical Center in the Bronx, New York, for respiratory symptoms due to PCR-confirmed COVID-19. Exclusion criteria were age < 21, history of prior SARS-CoV-2 infection, and/or underlying severe chronic lung diseases requiring home O2 and/or high dose steroids. We stratified and compared patients by whether they developed worsening respiratory failure, necessitating transfer to the intensive care unit (ICU) during their hospital stay. Using a custom Luminex Assay, we measured hospital admission serum concentrations of 8 host proteins, representing respiratory-associated epithelial (RAGE, SP-D, CC16), endothelial (Ang-2, vWF), and immune pathways (S100A12, ICAM-1, VCAM-1). Results. Except for race and WHO COVID-19 scores, demographics, co-morbidities, symptoms, and symptom duration were not statistically significantly different between patients requiring transfer to the ICU (n=15) and non-ICU patients (n=19). Higher log-transformed levels for 5/8 proteins (S100A12, ICAM-1, Ang-2, RAGE, SP-D) showed significant or marginally significant increased cause-specific hazard for ICU transfer (n=15). Estimated cumulative incidence functions further showed a significantly or near significantly increased risk for ICU transfer for patients with above the median values of S100A12 or ICAM-1 (p=0.013), Ang-2 (p=0.056) or RAGE (p=0.077), respectively (Figure 1). Host proteins predicting need for ICU transfer did not correlate strongly with other clinical laboratory markers for COVID-19 severity (CRP, LDH, D-Dimer, Fibrinogen, Ferritin). Comparison of estimated cumulative incidence at 7 days post admission for host protein markers above and below median levels for (A) S10012 (median 96,675 pg/ml);(B) ICAM-1 (median (1,192,277 pg/ml);(C) Ang-2 (median 3463 pg/ml);(D) RAGE (median 6356 pg/ml);and (E) SP-D (median 11,832 pg/ml). Conclusion. These results suggest that host proteins have additional predictive value for the severity of COVID-19-associated lung damage at time of presentation to the hospital.

Eicosapentaenoic acid favorably modulates protein expression in pulmonary endothelial inflammation

INTRODUCTION: SARS-CoV-2 and other viruses can cause endothelial cell (EC) dysfunction in multiple vascular beds, including pulmonary tissue. Infected patients may then develop acute respiratory distress syndrome (ARDS) and cardiovascular (CV) complications. The omega-3 fatty acid eicosapentaenoic acid (EPA) and its bioactive metabolites favorably modulate inflammation and EC function. These benefits of EPA may contribute to reduced CV events as reported in outcome trials (REDUCE-IT). Currently, EPA is being tested in patients with or at risk for COVID-19. This study tested the effects of either EPA pre- or post-treatment on global protein expression in human pulmonary ECs under conditions of inflammation using the cytokine IL-6 to simulate conditions of advanced viral infections. METHODS: Human lung microvascular endothelial cells (HMVEC-L) were pre-treated with either EPA (40 μM) or IL-6 (12 ng/mL) for 2 hr and then treated with IL-6 or EPA, respectively, for 24 hr in media with 2% FBS. Proteomic analysis was performed using LC/MS to assess relative protein expression levels. Only significant (p< 0.05) changes in protein expression between treatment groups >1-fold were analyzed. Expression of soluble intercellular adhesion molecule-1 (sICAM-1) was separately measured with immunochemistry. RESULTS: HMVEC-L pre- and post-treated with EPA during challenge with IL-6 showed significant changes in 100 (49/51 up/down) and 441 (229/212 up/down) proteins, respectively, compared with IL-6 treatment alone. Among the 31 proteins that were significantly modulated by both EPA pre- and post-treatment, thioredoxin reductase 1 increased relative to IL-6 alone, while matrix metalloproteinase 1 and fibronectin both decreased. Other proteins, such as hypoxia up-regulated protein 1, were differentially modulated by EPA relative to IL-6 (increased in pre-treatment, decreased in post-treatment). Finally, EPA significantly reduced sICAM- 1expression by 41% and 12% compared with IL-6 alone in the pre- and post-treatment models, respectively. CONCLUSIONS: These findings indicate that EPA favorably modulates the expression of multiple inflammatory and cytoprotective proteins during inflammation. These studies support a broad anti-inflammatory effect of EPA on pulmonary ECs that may have therapeutic implications for patients at risk for ARDS due to infectious agents including SARS-CoV-2 or other viruses.

COVID-19 Is Associated with Endothelial Dysfunction and Enhanced Plasma Thrombin Generation Despite Pharmacological Thromboprophylaxis

Background Hospitalised patients with severe COVID-19 (requiring critical care level support) appear to be at increased risk of thrombosis despite standard pharmacological thromboprophylaxis. The magnitude of thrombotic risk in patients with COVID-19 of moderate severity (not requiring critical care) is less clear. The optimal approach to thromboprophylaxis (and the role of intensified thromboprophylaxis) remains to be determined. Evidence of endothelial dysfunction has been widely reported in COVID-19 (particularly in severe COVID) and this may contribute to hypercoagulability. Aim To assess differences in patterns of hypercoagulability and endothelial dysfunction between a group of patients with moderate COVID-19 and a group of age-matched hospitalized patients (SARS-CoV-2 PCR negative) receiving low molecular weight heparin (LMWH) thromboprophylaxis. Methods Blood was collected from individuals admitted to hospital with COVID-19 of moderate severity (not requiring critical care level support) and a group of age-matched patients admitted with infective/inflammatory illness (SARS-CoV-2 PCR negative). All subjects received standard-dose LMWH thromboprophylaxis, with blood drawn at 12 hours post-dose (and with measurement of anti-FXa activity levels). Circulating levels of endothelial & fibrinolytic markers including ICAM, PAI-1, VCAM, soluble thrombomodulin (sTM), and tissue plasminogen activator (tPA) were determined by ELISA. Thrombin generation (TG) in platelet-poor plasma was assessed by calibrated automated thrombography in the presence of tissue factor (Final concentration, 1pM & 5pM), thrombomodulin (TM) (Final concentration, 6.25nM), and an inhibitory anti-tissue factor pathway inhibitor antibody (anti-TFPI;Final concentration 100μg/mL). Results 14 COVID-19 positive subjects and 11 hospitalized controls were recruited. There were no differences in mean age (69.7±4.5 vs 61.6±4.7 years;p= 0.2) or mean Body mass index (25.7±1.1 vs 22.7±1.2 Kg/m2;p=0.1) between groups. No COVID-19 patient or control required critical care support. In the COVID group, radiological evidence of pneumonitis [diffuse (n=3) or peripheral infiltrates (n=7)] was present in the majority of cases. None of the COVID-19 cases were requiring supplemental oxygen at the time of recruitment. All controls were admitted with either respiratory or urinary infection [radiological evidence of pneumonia in 4/11;supplemental oxygen requirement in 2/11, (28-36% FiO2 via nasal cannula)]. Plasma levels of sTM, ICAM, PAI-1 & VCAM were similar in both groups. Levels of t-PA were significantly higher in the COVID group (8.31±4.35 vs 4.91±2.37 ng/mL;p= 0.005). Despite similar plasma anti-Xa activity in both groups (0.06 vs 0.04 IU/mL;p=0.2), mean endogenous thrombin potential (ETP) was significantly higher in the COVID group (1929±119.7 vs 1528±138.9 nM*min;p=0.02), although peak thrombin was similar (173.6±26 vs 161.5±31nM). ETP-TM ratio was similar between groups (0.3±0.1 vs 0.2±0.1;p=0.3). Despite increased ETP, the lag time to thrombin generation was significantly prolonged in the COVID group (8.3±0.6 vs 5.8±0.5 mins, p= 0.006). This pattern has previously been observed in vascular diseases associated with altered plasma tissue factor pathway inhibitor (TFPI) activity. In the presence of an anti-TFPI antibody, the difference in lagtime between groups was attenuated (4.7±0.2 vs 3.5±0.1 mins;p= 0.002) and the difference in overall thrombin generation (delta TG) between both groups became significantly increased (Fig.1). Conclusion Plasma thrombin generation is enhanced in patients with non-severe COVID-19 despite pharmacological thromboprophylaxis. Endothelial dysfunction is also observed in this group and appears to modulate parameters of plasma thrombin generation. The clinical implications of these observations are not known although clinical studies of intensified thromboprophylaxis in attenuating thrombotic risk and other complications are ongoing. Fig 1. Inhibition of TFPI activity enhances thrombin generation in COVID-19. n the presence of an inhibitory anti-TFPI antibody, peak plasma thrombin generation was enhanced in COVID-19 in contrast to that observed among SARS-CoV-2 PCR negative hospitalised patients (339.6+25.2 vs 247.4+10.1, p=0.01). [Formula presented] Disclosures: Maguire: Actelion: Research Funding;Bayer Pharma: Research Funding. Ni Ainle: Daiichi-Sankyo: Research Funding;Actelion: Research Funding;Leo Pharma: Research Funding;Bayer Pharma: Research Funding. Kevane: Leo Pharma: Research Funding.

Hyperbaric oxygen therapy and its suitability as an adjuvant for treatment of covid-19 patients: A review

The outbreak of the SARS CoV2 ’Coronavirus pandemic’ is believed to have originated in Wuhan in 2019 as a zoonotic spread from bats to humans. It is a highly communicable infection-causing rapid human to human transmission of the virus by virtue of its infectious and pleomorphic nature. The virus has affected millions of people worldwide, with numbers still rising with each passing day. Depleting oxygen saturation levels is amongst the prime concerns in the majority of infected patients. Nasal prongs, face masks, mechanical ventilation and extracorporeal membrane (ECMO) are the commonly used modes of oxygen delivery in such patients. These methods though mostly successful, at times fail to restore the depleting oxygen levels to normal. Hyperbaric oxygen therapy (HBOT) involves the administration of 100% O2 in a special chamber whose pressure is maintained at a level greater than 1 ATP. The main purpose for raising the pressure within the chamber is that as the atmospheric pressure increases, the saturation levels of oxygen in the blood also increase, which eventually result in increased overall tissue oxygenation. This article provides a systematic and wholesome review on the basic principle of hyperbaric oxygen therapy, its effects on the body at a microscopic and macroscopic level, its various uses and its suitability as an adjuvant for the treatment of select COVID-19 infected patients.

Elevated plasma ICAM1 levels predict 28-day mortality in cirrhotic patients with COVID-19 or bacterial sepsis.

BACKGROUND & AIMS: Endothelial injury and dysfunction play a detrimental role in the pathogenesis of infections. Endothelium-related molecules have been reported as potential diagnostic and/or prognostic biomarkers of infection. The prognostic value of these biomarkers in patients with cirrhosis and infections remains elusive. METHODS: In this study, we investigated the performance of key soluble endothelial injury biomarkers, including intercellular adhesion molecule 1 (ICAM1), von Willebrand factor (vWF), vascular endothelial growth factor receptor 1 (VEGFR1), and angiopoietin 1 and 2 (Ang1, 2) as mortality predictors in patients with cirrhosis and severe COVID-19 or bacterial sepsis. RESULTS: A total of 66 hospitalized patients (admitted to the COVID-19 ward or liver intensive care unit [ICU]) were included. Twenty-two patients had COVID-19 alone, while 20 patients had cirrhosis plus COVID-19. Twenty-four patients had cirrhosis plus bacterial sepsis. Among patients with cirrhosis, the most common aetiology of liver disease was alcohol. ICAM1 was increased (p = 0.003) while VEGFR1 (p <0.0001) and Ang1 (p <0.0001) were reduced in patients with COVID-19 and cirrhosis, compared to patients with COVID-19 alone. Endothelial biomarker levels did not differ significantly between patients with cirrhosis and severe COVID-19 or bacterial sepsis in the ICU. In these patients, ICAM1 levels significantly and independently predicted mortality (hazard ratio 3.24; 95% CI 1.19-8.86) along with model for end-stage liver disease (MELD) score, renal and coagulation failures. The AUC for ICAM1 was 0.74, MELD was 0.60 and combined ICAM1 and MELD was 0.70. ICAM1 also positively correlated with the composite organ failure scores recorded 3-5 days post ICU admission (CLIF-OF and SOFA) in this subgroup of patients. CONCLUSION: The study indicates that in patients with cirrhosis, elevated plasma ICAM1 serves as an independent predictor of severe COVID-19- or sepsis-associated 28-day mortality. LAY SUMMARY: Bacterial sepsis and COVID-19 lead to increased mortality in patients with cirrhosis. In this study, we demonstrate that high plasma levels of ICAM1, an endothelial injury biomarker, is one of the important factors predicting mortality in critically ill cirrhotic patients with severe COVID-19 or bacterial sepsis.

Markers of endothelial and epithelial pulmonary injury in mechanically ventilated COVID-19 ICU patients.

BACKGROUND: Biomarkers can be used to detect the presence of endothelial and/or alveolar epithelial injuries in case of ARDS. Angiopoietin-2 (Ang-2), soluble intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion protein-1 (VCAM-1), P-selectin and E-selectin are biomarkers of endothelial injury, whereas the receptor for advanced glycation end-products (RAGE) reflects alveolar epithelial injury. The aims of this study were to evaluate whether the plasma concentration of the above-mentioned biomarkers was different 1) in survivors and non-survivors of COVID-19-related ARDS and 2) in COVID-19-related and classical ARDS. METHODS: This prospective study was performed in two COVID-19-dedicated Intensive Care Units (ICU) and one non-COVID-19 ICU at Ferrara University Hospital. A cohort of 31 mechanically ventilated patients with COVID-19 ARDS and a cohort of 11 patients with classical ARDS were enrolled. Ang-2, ICAM-1, VCAM-1, P-selectin, E-selectin and RAGE were determined with a bead-based multiplex immunoassay at three time points: inclusion in the study (T1), after 7 ± 2 days (T2) and 14 ± 2 days (T3). The primary outcome was to evaluate the plasma trend of the biomarker levels in survivors and non-survivors. The secondary outcome was to evaluate the differences in respiratory mechanics variables and gas exchanges between survivors and non-survivors. Furthermore, we compared the plasma levels of the biomarkers at T1 in patients with COVID-19-related ARDS and classical ARDS. RESULTS: In COVID-19-related ARDS, the plasma levels of Ang-2 and ICAM-1 at T1 were statistically higher in non-survivors than survivors, (p = 0.04 and p = 0.03, respectively), whereas those of P-selectin, E-selectin and RAGE did not differ. Ang-2 and ICAM-1 at T1 were predictors of mortality (AUROC 0.650 and 0.717, respectively). At T1, RAGE and P-selectin levels were higher in classical ARDS than in COVID-19-related ARDS. Ang-2, ICAM-1 and E-selectin were lower in classical ARDS than in COVID-19-related ARDS (all p < 0.001). CONCLUSIONS: COVID-19 ARDS is characterized by an early pulmonary endothelial injury, as detected by Ang-2 and ICAM-1. COVID-19 ARDS and classical ARDS exhibited a different expression of biomarkers, suggesting different pathological pathways. Trial registration NCT04343053 , Date of registration: April 13, 2020.