Histomorphological Spectrum of Lung Lesions at Autopsy-A Tertiary Care Centre Experience

Background and aims: A wide variety of pathological conditions involve the lungs. In autopsy, the lungs are examined for disease, injury and other findings suggesting cause of death or related changes.Aims & Objectives: The present study aimed to study the histomorphological spectrum of lung lesions at autopsy and to assess the frequency of different types of lesions;and to associate histomorphological changes with cause of death.Material and Methods: It was a one-year observational study conducted in the Department of Pathology, Govt. Medical College, Jammu. Lung tissue pieces from all medicolegal autopsies received were fixed, examined grossly, processed;paraffin embedded sections obtained were stained with Hematoxylin and Eosin stain and examined under microscope. Findings were recorded and tabulated. Result(s): Out of 264 cases, males were predominantly affected (84%);median age was 38 years. The various changes observed were congestion (68%), edema (45.4%), pneumonia (5%), granulomatous inflammation (3%), diffuse alveolar damage (1.5%), haemorrhage (14.4%), interstitial changes (60%), malaria (0.4%) and malignancy (0.4%). Natural deaths were the commonest cause (75, 28%) followed by asphyxial deaths (65, 24.6%). Conclusion(s): Histopathological examination of lung autopsies highlights many incidental findings, establishes underlying cause of death, serves as a learning tool and also holds scope for detection of newer diseases.Copyright © 2023 JK Science.

FEATURES OF IMMUNOHISTOCHEMICAL MARKERS OF STEM CELLS CD-117, CD-44 IN FEMALE PLACENTAS ON THE BACKGROUND OF CHRONIC STRESS.

Background. Placenta is a subject of interest to a wide range of scientists because it is rich in stem cells and their precursors. A stem cell is a cell that has the ability to self-repair and can differentiate into offspring (daughter cells) of one or more germ layers. In recent years, scientists have obtained new data of stem cells regenerative potential. However, only isolated publications about placental stem cells are available. Therefore, our studies about placental stem cells are important for discovery of structural and molecular mechanisms, their changes under the influence of chronic stress. Objective(s): to study the features of immunohistochemical markers of pluripotent stem cells and their morphological features. Materials and methods. We examined 80 women placentas with chronic stress in comparison with control using general histological and immunohistochemical methods in the following groups: group 1 - women placentas with physiological course of pregnancy in term 38-40 weeks, group 2 - women placentas with miscarriage, group 3 - women placentas with chronic stress due to internal irradiation (4.5 Bq/kg and more), group 4 - women placentas which had COVID-19 during pregnancy. Results. There was a significant increase of stem cell markers expression in the three study groups with a significant predominance in groups 3 and 4. It was also determined the different direction of their active factors. Conclusions. The general changes of all structures of the placental barrier are detected as a result of chronic stress due to various factors: micro detachment of the decidual membrane (significant increase in cases in the studied groups);malperfusion in the structures of the maternal placental barrier;in the placenta stem cells of the three study groups in comparison with the control were found stress markers. Thus, chronic stress due to various factors causes the same type of changes in placental structures, but they have different degrees of expression - with internal irradiation doses >= 4.8 Bq/kg, these changes are most expressive.Copyright © 2022 Authors. All rights reserved.

PATHOMORPHOLOGICAL FEATURES OF ACUTE RESPIRATORY DISTRESS SYNDROME IN COMPETING LUNG DISEASES: COVID-19 AND SARCOIDOSIS.

The COVID-19 pandemic is a worldwide problem. The clinical spectrum of SARS-CoV-2 infection varies from asymptomatic or paucity-symptomatic forms to conditions such as pneumonia, acute respiratory distress syndrome and multiple organ failure. Objective was to describe a clinical case of SARS-CoV-2 infection in the patient with sarcoidosis and cardiovascular pathology developing acute respiratory syndrome and lung edema. Material and methods. There were analyzed accompanying medical documentation (outpatient chart, medical history), clinical and morphological histology data (description of macro- and micro-preparations) using hematoxylin and eosin staining. Results. Lung histological examination revealed signs of diffuse alveolar damage such as hyaline membranes lining and following the contours of the alveolar walls. Areas of necrosis and desquamation of the alveolar epithelium in the form of scattered cells or layers, areas of hemorrhages and hemosiderophages are detected in the alveolar walls. In the lumen of the alveoli, a sloughed epithelium with a hemorrhagic component, few multinucleated cells, macrophages, protein masses, and accumulated edematous fluid were determined. Pulmonary vessels are moderately full-blooded, surrounded by perivascular infiltrates. Signs of lung sarcoidosis were revealed. Histological examination found epithelioid cell granulomas consisting of mononuclear phagocytes and lymphocytes, without signs of necrosis. Granulomas with a proliferative component and hemorrhage sites were determined. Giant cells with cytoplasmic inclusions were detected - asteroid corpuscles and Schauman corpuscles. Non-caseous granulomas consisting of clusters of epithelioid histiocytes and giant Langhans cells surrounded by lymphocytes were detected in the lymph nodes of the lung roots. Hamazaki-Wesenberg corpuscles inside giant cells were found in the zones of peripheral sinuses of lymph nodes. In the lumen of the bronchi, there was found fully exfoliated epithelium, mucus. Granulomas are mainly observed subendothelially on the mucous membrane, without caseous necrosis. Histological examination of the cardiovascular system revealed fragmentation of some cardiomyocytes, cardiomyocyte focal hypertrophy along with moderate interstitial edema, erythrocyte sludge. Zones of small focal sclerosis were determined. The vessels of the microcirculatory bed are anemic, with hypertrophy of the walls in small arteries and arterioles. Virological examination of the sectional material in the lungs revealed SARS-CoV-2 RNA. Conclusion. Based on the data of medical documentation and the results of a post-mortem examination, it follows that the cause of death of the patient R.A., 50 years old, was a new coronavirus infection COVID-19 that resulted in bilateral total viral pneumonia. So-morbidity with competing diseases such as lung sarcoidosis and cardiovascular diseases aggravated the disease course, led to the development of early ARDS and affected the lethal outcome. Copyright © 2022 Saint Petersburg Pasteur Institute. All rights reserved.

COVID-19 Vaccination-Induced Cholangiopathy and Autoimmune Hepatitis: A Series of Two Cases.

The coronavirus disease 2019 (COVID-19) pandemic has been associated with significant morbidity and mortality. Following the introduction of vaccines, various side effects have been reported. Whilst those reported may be attributed to the vaccine itself, at times, it may simply incite an immunological phenomenon. We present a case series of two patients who presented with symptoms of yellowing of the eyes and the skin along with fatigue, and tiredness, following vaccination for COVID-19. The diagnosis of post COVID-19-vaccination related hepatitis is one of the fewer, less understood, yet reported side effects associated with significant morbidity. The diagnosis of COVID-19 vaccination-related cholangitis is an outcome reported here for the first time to the best of our knowledge. It was alarming that both patients did not have any significant past history of medical ailments. A prompt assessment followed by investigations including liver biopsy assisted in a timely understanding of the phenomenon with complete resolution of the symptoms.

Blood identified and quantified in formalin fixed paraffin embedded lung sections using eosin fluorescence.

Eosin Y is a common stain in histology. Although usually used for colourimetric imaging where the dye is used to stain pink/red a range of structures in the tissue, Eosin Y is also a fluorochrome, and has been used in this manner for decades. In this study our aim was to investigate the fluorescence properties of the dye to enable quantification of structures within formalin-fixed paraffin-embedded (FFPE) tissue sections. To do this, FFPE sections of hamster tissue were prepared with haematoxylin and eosin Y dyes. Spectral detection on a confocal laser scanning microscope was used to obtain the fluorescence emission spectra of the eosin Y under blue light. This showed clear spectral differences between the red blood cells and congealed blood, compared to the rest of the section. The spectra were so distinct that it was possible to discern these in fluorescence and multi-photon microscopy. An image analysis algorithm was used to quantify the red blood cells. These analyses could have broad applications in histopathology where differentiation is required, such as the analysis of clotting disorders to haemorrhage or damage from infectious disease.

THE LINK between INFLAMMATION and THROMBOSIS: CLINICAL and IMMUNOHISTOCHEMICAL CHARACTERIZATION of PULMONARY ARTERIAL THROMBOSIS in COVID-19 PATIENTS

Background: Coronavirus-19 disease (COVID-19) has been responsible, to date, for more than 5 million of deaths. Immunothrombosis may be a major factor contributing to mortality in COVID-19 and pulmonary arterial tree involvement that mimics multiple pulmonary embolism could be a major contributor to disease course. Immunomodulatory drugs are of some beneft but mechanism not completely clear. We investigated pulmonary arterial tree clots to better appreciate their immunothrombotic nature, in contrast to the pathological characteristics of non-infammatory thrombi (1). Objectives: The primary objective was to study in depth the arterial thrombosis in COVID-19, by characterizing the immunohistochemical nature of thrombi, performing macroscopic and microscopic analyses, and by comparing clinical, laboratory and anatomical-pathological data of these patients with other patients died for COVID-19 but without evidence of pulmonary arterial thrombosis. Methods: Autopsies were performed in patients (cases) who died for COVID-19 with evidence of pulmonary arterial thrombosis at autopsy fnding but without pathological signs of bronchopneumonia or peripheral venous thrombosis. COVID-19 positive patients without pulmonary arterial thrombosis were selected as control group. Hematoxylin and eosin stained slides were reviewed choosing those with visible pulmonary thrombi. Further histochemical and immunohisto-chemical staining were performed in selected paraffin blocks. Each component of the thrombus was evaluated with the software application QuPath in terms of fbrin, red blood cells, platelets and immune cells percentage after scanning the slides with Aperio System. Laboratory tests were recorded at 2 points: at hospital admission and at Intensive Care Unit transfer. Results: We included 13 patients (cases) and 14 controls, matched for age, gender and time from diagnosis to death. Twenty arterial thrombi were studied. By immuno-histochemistry, arterial thrombi were composed by white blood cells (WBC) [median, IQR range: 10% (5-12.25)], mainly neutrophils [58% (35.2-64.5)], red blood cells [12%, (6-34.25)], fbrin [19% (14.5-42.25)], platelets [39%, (31.75-48)] (Figure 1). Three cases had a history of previous thrombosis. All cases had received anticoagulant treatment during hospitalization, low molecular weight heparin in 12/13 (therapeutic regimen in 4/12, prophylactic in 8/12) while 1/13 continued oral anticoagulants for comorbidity. By comparing laboratory fndings between cases and controls, cases showed signifcantly higher levels of platelet count [median, IQR range: 195000/mmc (157750-274500) vs 143500 (113000-175250), p=0.011], LDH [854 U/l (731-1315) vs 539 (391.5-660), p=0.003)] at hospital admission, and D-dimer at ICU transfer [25072 FEU (6951-50531) vs 1024 (620-5501), p=0.003)]. Conclusion: Pulmonary arterial thrombosis in COVID-19 is a type of immune-mediated infammatory thrombosis, since the amount of WBC is 6-times more than normal value seen in non-infammatory thrombi. Some markers of infammation, necrosis and coagulation are much more increased in this subset of patients. Chest CT angiography rather than simple CT scan at hospital admission could be more useful in this setting, and treatments with antiplatelet agents or anticoagulants, eventually in combination with immunotherapy, might positively affect the outcome.

Efficacy of Probiotic on Duodenal TNF-α Expression and the Histological Findings in the Liver and Lung in Animal Model Canine Coronavirus

Currently, Canine coronavirus (CCoV) is an enteric pathogen of the Alphacoronavirus-1 species that causes mild to severe diarrhea in puppies. The pathogenesis of this infection will cause severe lymphopenia and lead to death in puppies. This study aimed to determine the administration of probiotics on TNF-α expression, histological findings of the liver and lung in mice infected with CCoV. A total of 28 mice were randomly assigned into seven treatment groups, i.e. (C-) placebo;(C+) active CCoV vaccine induction;(T1) CCov + Isopronosin;(T2) CCoV + Lactobacillus acidophilus probiotic;(T3) CCoV + Lactobacillus Acidophylus and Bifidobacterium probiotics;(T4) CCoV + colustrum fermentation probiotic;(T5) CCoV + ginger, turmeric and ginger probiotics. Thereafter, the expression of TNF-α in the duodenum was stained using immunohistochemistry, liver and lung were stained using hematoxylin eosin. The data were analyzed using the ANOVA test followed by the Tukey test with a significance level (p<0.05). TNF-α expression on T4 and T5 decreased significantly (p<0.05) compared to C+, T1, T2 and T3. Histologic findings of the liver in the C- and T4 groups showed normal features in the central vein. On the other hand, glycogen accumulation was found in hepatocyte cells, hemorrhage with sinusoid dilation, lymphocyte infiltration in centro lobular area in group C+. Lung histology showed normal features of sinusoids and alveolar septa in groups C- and T4. Meanwhile, intra-alveolar hemorrhage was found with neutrophil cell infiltration and fibrin plasma accumulation in group C+. In conclusion, colostrum fermentation probiotics can reduce TNF-α expression in the duodenum and improve the liver and lung physiology in mice infected with CCoV.

SARS-COV-2 ENDOTHELIAL DYSFUNCTION CAN CAUSE ERECTILE DYSFUNCTION: HISTOPATHOLOGICAL, IMMUNOHISTOCHEMICAL AND ULTRASTRUCTURAL STUDY OF THE HUMAN PENIS

OBJECTIVE: To describe the histopathological features of penile tissue of patients who recovered from symptomatic COVID-19 infection and subsequently developed severe erectile dysfunction (ED). MATERIALS AND METHODS: After providing informed consent, penile tissue was collected from patients undergoing surgery for inflatable penile prosthesis due to severe ED under an IRB approved protocol. Two specimens were obtained from men with a history of COVID-19 infection and two specimens were obtained from men with no history of infection (all men tested negative immediately before surgery). Tissue from COVID-19 (+) and COVID-19 (-) specimens were imaged with transmission electron microscopy (TEM). The tissue was analyzed for viral RNA using polymerase chain reaction (PCR) and viral spike protein. Formalin-fixed paraffinembedded tissues were stained with hematoxylin and eosin (H&E) and subjected to immunohistochemical analysis for endothelial Nitric Oxide Synthase (eNOS) expression (marker of endothelial function). Endothelial progenitor cells (EPC) function was assessed ex vivo by determination of endothelial colony forming units from blood samples collected from the COVID-19 (+) and COVID (-) men with severe ED. RESULTS: TEM revealed extracellular viral particles ∼100 nm in diameter, with prominent peplomers (spikes), and electron-dense dots of the nucleocapsid inside the particles near penile vascular endothelial cells of the COVID-19 (+) patients. Notably, viral particles were not detected in tissue obtained from COVID-19 (-) men. COVID-19 RNA was detected in both the penis biopsy samples from men with a history of COVID, but not in the samples from COVID-19 (-) men. There were no significant differences in H&E staining between COVID-19 (+) and COVID-19 (-) men and viral spike protein was not detected. Immunohistochemistry showed decreased eNOS expression in the corpus cavernosum of COVID-19 (+) men compared to COVID-19 (-) men, consistent with endothelial dysfunction. COVID-19 spike protein-positive cells could not be detected by immunofluorescence despite positive COVID-19 PCR. EPC levels from the COVID-19 (+) men were 0 cell/well and 1.167 cell/well respectively compared to mean EPCs from 34 COVID-19 (-) men with severe ED (4.04 cells/well), suggesting impaired endothelial function. CONCLUSIONS: Our study is the first to demonstrate the presence of COVID-19 virus in the penis long after the initial infection in humans. Our study also suggests that widespread endothelial cell dysfunction from COVID-19 infection can contribute to resultant erectile dysfunction. Future studies will evaluate novel molecular mechanisms of how COVID-19 infection leads to ED. IMPACT STATEMENT: COVID-19 can linger in the penis long after initial infection and can contribute to erectile dysfunction.

COVID-19 Patients with GI Manifestations May be Associated with Extensive Microthrombosis in the Small Intestine, a Study from 13 Autopsy Cases

Background: More than 20% of COVID-19 patients have gastrointestinal (GI) symptoms, among which diarrhea is the most commonly seen symptom. Studies have suggested that patients with severe disease are more likely to have abdominal manifestations. Recent studies have also implicated that coagulopathy and thromboembolic as the major pathophysiological event leading to higher mortality. Besides thrombus in larger vessels, microthrombi appears to occur systemically and plays an important role in multiple organ dysfunction. However, fewer studies have focused on microthrombosis in the GI system. Design: 13 bronchial SARS-CoV-2 PCR proven autopsy cases were included in the study. Small intestinal specimens were obtained, and processed to routine hematoxylin and eosin (H&E) and CD61 immunohistochemistry (IHC). Related clinical and laboratory data were collected from patient chats. H&E and IHC slides were reviewed by two GI pathologists to evaluate histopathology and microthrombi. The degree of microthrombosis was graded as no microthrombi, focal (1- 2 per 10x), scattered (3-5 per 10x), and diffuse (≥6 per 10x). Results: Out of 13 patients (11 males, 1 female, age range 22-89 years old), 6 had diarrhea as the initial GI symptom, while others (7 patients) did not report any GI manifestations. Sections from the small intestine showed no acute inflammation in all cases. CD61 positive microthrombi was seen in all small intestine specimens, mainly located in the microvasculature of mucosa, and occasionally submucosal tissue. Patients who had diarrhea, 4/6 (66.7%) showed diffuse (greater than 6 per 10x field) microthrombi in the small intestine. In contrast, patients without GI symptoms, only 2/7 (28.5%) had diffuse microthrombi. Data from lab tests showed the D-dimer appeared to be higher in patients with diarrhea (median, 4067, range from 867 to 10000 ng/ml) compared to patients without diarrhea (median, 2820, range from 298 to 10000 ng/ml). There was no significant difference between median levels of C-reactive protein, prothrombin time (PT) and partial thromboplastin time (aPTT) between patients with or without diarrhea. Conclusions: Our study highlights that microthrombi frequently occurs in the GI system as reported in other organs. COVID-19 patients with initial GI manifestations, may develop severe microthrombosis and progress to sever disease.

Cranial nerve involvement in mucormycosis in post-COVID patients: a case series

Background: One of the largest outbreaks of rhinosinocerebral mucormycosis (RSCM) occurred in India close to the second wave of the SARS-CoV-2 infection. RSCM is a rare infection caused by several fungal species occurring in immunocompromised subjects. Mucor shows a high propensity to invade the central nervous system. There have been limited studies, mostly isolated case reports, on the neurological manifestations of RSCM. The outbreak of mucormycosis infection was thus the most opportune to study the neurological manifestations and cranial nerve involvement in mucormycosis in greater depths. Aim of the study: The purpose of the study was to investigate and review the involvement of cranial nerves in a series of cases of rhinosinocerebral mucormycosis associated with the novel coronavirus disease caused by SARS-CoV-2. Results: It was a retrospective cross-sectional study of seven patients who were undergoing treatment of RSCM with a recent history of coronavirus disease caused by SARS-CoV-2 infection within the last 3 months. Patients with cranial nerve involvement were identified by magnetic resonance imaging (MRI) at a single institution. Demographic details of the patients, clinical presentation, imaging, microbiological and pathological findings were recorded. All subjects had two or more cranial nerves affected by fungal infection. The most commonly involved cranial nerve was found to be the optic nerve followed by the trigeminal nerve and its branches. We document three cases with extensive involvement of the inferior alveolar branch of the mandibular division of the trigeminal nerve (V3), a previously unreported finding. In one case, in addition to the second and fifth cranial nerves, the third, fourth, sixth, seventh, eighth, and twelfth cranial nerves were involved without any sensory or motor long tract involvement, suggestive of Garcin syndrome secondary to intracranial abscesses and skull base osteomyelitis due to invasive fungal infection. This case is of rare occurrence in the literature, and our study provides one such example. Conclusion: Cranial nerve involvement in patients of mucormycosis tends to have a poor prognosis, both cosmetic and functional. Radical surgeries and aggressive medical management is needed in such cases to improve the outcome.

Activation of immune system may cause pathophysiological changes in the myocardium of sarscov-2 infected macaque model

Background: Recent reports suggest the presence of the SARS-CoV-2 virus in the myocardium of patients who died from the COVID-19 disease. Cardiovascular injury in COVID-19 patients is an established extra-pulmonary manifestation of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection which may lead to induction of arrhythmia, acute heartfailure, thickening of ventricular wall, reduced ejection fraction and thromboembolism. Non-human primates (NHP) provide a useful model to study cardiovascular changes due to their homology to the ACE2 receptor in humans. Aim: The aim of this study is to characterize the pathological changes in the heart of SARS-CoV-2 infected NHPs. Methods: In the present study, SARS-CoV-2 infected primates via aerosol route (n=4), multi-routes (i.e., oral, nasal, intratracheal and conjunctival) (n=4), and a control group (n=5) were included. Heart tissue samples were collected and the left ventricular tissue was analyzed by hematoxylin and eosin, trichrome, and immunohistochemical staining specific to CD3, CD68 andSARS-CoV-2 nucleocapsid protein.Results: Several pathological findings were observed in the heart, including cardiomyocyte disarray, mononuclear infiltrates of inflammatory cells as well as hypertrophy. Collagen specific staining showed development of cardiac fibrosis in the interstitial as well as in the perivascularregion in the hearts of infected primates. Moreover, the myocardial tissue samples displayed multiple foci of inflammatory cells positive for T lymphocytes and macrophages within the myocardium. Additionally, SARS-CoV-2 nucleocapsid protein staining detected the presence of virus particles in the myocardium. Conclusion: COVID19 infection is characterized by exaggerated inflammatory immune response in the heart which possibly contributes to myocardial remodeling and subsequent fibrosis. These findings suggest progression of disease which could lead to development of severe complications including heart failure.

Susceptibility of mature human myocardial cell types to sars-cov-2

Background: SARS-CoV-2 is a highly transmissible and virulent respiratory pathogen responsible for the global coronavirus disease 2019 (COVID-19) pandemic. A significant number of patients infected with SARS-CoV-2 show signs of myocardial injury ranging from asymptomatic troponemia to acute congestive heart failure and cardiogenic shock. The precise mechanisms underlying myocardial injury in this cohort are unclear, and it is difficult to distinguish weather new onset cardiac dysfunction is representative of active myocardial infection or a consequence of systemic illness. To address this gap in knowledge we constructed a model to assess replicative potential of SARS-CoV2 in primary cell lines derived from adult and pediatric myocardium including cardiomyocytes, fibroblasts, and endothelial cells and corroborated our in vitro findings with a pathologic analysis of myocardial tissue obtained from patients infected with SARS-CoV-2. Methods: Samples of atrial myocardium obtained from patients undergoing cardiac surgery were enzymatically digested and purified into cardiomyocyte, fibroblast, and endothelial cell populations. Susceptibility to infection with SARS-CoV-2 was then assessed in primary human myocardial cell types and compared against induced cardiomyocytes derived from human pluripotential stem cells. Infectivity was quantitatively assessed using qPCR against genomic and subgenomic viral RNA and normalized to GAPDH. Postmortem heart and lung FFPE tissue from de-identified patients who died from SARS CoV-2 infection were obtained and analyze by immunofluorescence for viral spike and nucleocapsid protein or stained with hematoxylin and eosin for histological evaluation. Results: Primary cardiomyocytes obtained from adult (n=7) and pediatric (n=7) atrial myocardium could not support active replication SARS-CoV-2 virus and there was no evidence of viral replication in pathologic myocardial specimens obtained from COVID infected patients (n=7). Collectively our data indicate that primary cardiac cell types are unable to support the level of viral replication observed in iPSCM (p=0.0007) suggesting that induced pluripotential stem cells may not adequately model the response of mature myocardium to SARS-CoV-2.

Inhibition of Sars-Cov-2 Viral Replication and In Vivo Thrombus Formation By a Novel Plant Flavonoid

Plant-based flavonoids have been examined as inhibitors of β-coronavirus replication and as potential therapeutics for COVID19 based on their safety profile and widespread availability. SARS-CoV-2 viral replication is dependent on a cysteine protease known as 3CL protease, or main protease (Mpro), which cleaves the polyprotein translated from SARS-CoV-2 ssRNA into 11 functional proteins. This protease is highly conserved among β-coronaviruses and is intolerant of mutation. The main protein (Mpro) of SARS-CoV, SARS-CoV-2, and MERS has been identified as a target of flavonoids both by in silico and in vitro approaches. We have previously showed that select flavonoids inhibit protein disulfide isomerase (PDI), which is essential for normal thrombosis. These flavonoid PDI inhibitors block thrombus formation in vivo and have shown efficacy as antithrombotics in clinical studies. Given the substantial morbidity and mortality caused by COVID19-associated coagulopathy, we sought to identify a flavonoid that inhibits both SARS-CoV-2 Mpro and PDI, potentially blocking both viral replication and thrombus formation. While in silico studies identified many flavonoids as SARS-CoV-2 main protein (Mpro) inhibitors, no comprehensive in vitro testing of flavonoids against SARS-CoV-2 has previously been performed. We therefore evaluated 1,020 diverse flavonoids using high throughput screening for their ability to inhibit SARS-CoV-2 Mpro in a fluorescence-based Mpro substrate cleavage assay. This analysis identified four new flavonoid inhibitors of Mpro that had IC 50s ranging from 5-15 µM: amentoflavone, 3,8'-biapigenin, jaceidin triacetate, and pinocembrin 7-O-(3“-galloyl-4”,6“-(S)-hexahydroxydiphenoyl)-beta-D-glucose (PGHG). These compounds were equally or more potent than previously identified flavonoid inhibitors of SARS-CoV-2 Mpro, baicalein and myricetin. Structure activity relationships identified apigenin as an additional Mpro inhibitor. In a Vero-E6-based assay of SARS-CoV-2 replication, PGHG inhibited with an IC 50 = 4.9 µM. At 50 µM, apigenin showed 94±2.1% inhibition and baicalein 65±8.0% inhibition, while myricetin, amentoflavone, and 3,8'-biapigenin did not inhibit viral replication. Jaceidin triacetate was too toxic for further analysis. We next evaluated novel Mpro inhibitors for their ability to inhibit PDI. The most potent PDI inhibitor was PGHG, which blocked PDI reductase activity in an insulin turbidimetric assay with an IC 50 = 3.99±1.14 µM and in a di-eosin-GSSG assay with an IC 50 = 1.50±0.60 µM. When tested against isolated fragments of PDI, PGHG inhibited isolated a and a' fragments as well as ab, b'xa' and abb'x fragments, indicating that it acts on the a and a' domains of PDI. Since PDI is essential for thrombosis, we evaluated whether PGHG blocks platelet accumulation and fibrin formation following vascular injury. We infused mice with 25 mg/kg PGHG or vehicle and subsequently induced thrombus formation via laser-induced injury of an arteriole within the cremaster circulation. Infusion of PGHG resulted in a 82±6.2% inhibition of platelet accumulation and a 79±3.7% inhibition of fibrin formation. In contrast 25 mg/kg had no significant effect on tail bleeding in mice compared to vehicle control. Targeted therapies remain an important component of the armamentarium against COVID19. Our results show that a naturally occurring flavonoid, PGHG, found in Penthorum chinense Pursh, inhibits both SARS-CoV-2 replication and thrombosis without enhancing bleeding. This observation provides proof-of-principle for the development of plant-based flavonoid therapies for inhibition of β-coronaviruses and supports the further evaluation of PGHG for therapeutic use in COVID19. [Formula presented] Disclosures: No relevant conflicts of interest to declare.

The potential of BEN815 as an anti-inflammatory, antiviral and antioxidant agent for the treatment of COVID-19.

Background: The corona virus disease 2019 (COVID-19) pandemic has highlighted the fact that there are few effective antiviral agents for treating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Although the very recent development of vaccines is an extremely important breakthrough, it remains unclear how long-lived such vaccines will be. The development of new agents therefore remains an important goal. Purpose: Given the multifaceted pathology of COVID-19, a combinatorial formulation may provide an effective treatment. BEN815, a natural nutraceutical composed of extracts from guava leaves (Psidium guajava), green tea leaves (Camellia sinensis), and rose petals (Rosa hybrida), had previously shown to have a therapeutic effect on allergic rhinitis. We investigated whether BEN815 possesses anti-inflammatory, antiviral and antioxidant activities, since the combination of these effects could be useful for the treatment of COVID-19. Study design: We examined the anti-inflammatory effects of BEN815 and its principal active components quercetin and epigallocatechin gallate (EGCG) in lipopolysaccharide (LPS)-induced RAW264.7 cells and in an LPS-challenged mouse model of endotoxemia. We also assessed the antioxidant activity, and antiviral effect of BEN815, quercetin, and EGCG in SARS-CoV-2-infected Vero cells. Methods: The principal active ingredients in BEN815 were determined and quantified using HPLC. Changes in the levels of LPS-induced pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α were measured by ELISA. Changes in the expression levels of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) were analyzed using western blotting. Antioxidant assay was performed using DPPH and ABTS assay. SARS-CoV-2 replication was measured by immunofluorescence staining. Results: BEN815 significantly suppressed the induction of IL-6 and TNF-α as well as COX-2 and iNOS in LPS-induced RAW264.7 cells. In addition, BEN815 protected against LPS-challenged endotoxic shock in mice. Two major constituents of BEN815, quercetin and EGCG, reduced the induction of IL-6 and TNF-α as well as COX-2 and iNOS synthase in LPS-induced RAW264.7 cells. BEN815, quercetin, and EGCG were also found to have antioxidant effects. Importantly, BEN815 and EGCG could inhibit SARS-CoV-2 replications in Vero cells. Conclusion: BEN815 is an anti-inflammatory, antiviral, and antioxidant natural agent that can be used to prevent and improve inflammation-related diseases, COVID-19.

Histopathological features in fatal COVID-19 acute respiratory distress syndrome.

Background: COVID-19 acute respiratory distress syndrome (ARDS) shares the common histological hallmarks with other forms of ARDS. However, the chronology of the histological lesions has not been well established. Objective: To describe the chronological histopathological alterations in the lungs of patients with COVID-19 related ARDS. Design: A prospective cohort study was carried out. Setting: Intensive Care Unit of a tertiary hospital. Patients: The first 22 consecutive COVID-19 deaths. Measurements: Lung biopsies and histopathological analyses were performed in deceased patients with COVID-19 related ARDS. Clinical data and patient course were evaluated. Results: The median patient age was 66 [63-74] years; 73% were males. The median duration of mechanical ventilation was 17 [8-24] days. COVID-19 induced pulmonary injury was characterized by an exudative phase in the first week of the disease, followed by a proliferative/organizing phase in the second and third weeks, and finally an end-stage fibrosis phase after the third week. Viral RNA and proteins were detected in pneumocytes and macrophages in a very early stage of the disease, and were no longer detected after the second week. Limitation: Limited sample size. Conclusions: The chronological evolution of COVID-19 lung histopathological lesions seems to be similar to that seen in other forms of ARDS. In particular, lung lesions consistent with potentially corticosteroid-sensitive lesions are seen.

Antecedentes: El síndrome de dificultad respiratoria aguda (SDRA) asociado a la COVID-19 comparte características histológicas con otros tipos de SDRA. Sin embargo, no se ha establecido adecuadamente la cronología de las lesiones histológicas. Objetivo: Describir las alteraciones histopatológicas cronológicas en los pulmones de los pacientes con síndrome de dificultad respiratoria aguda asociado a COVID-19. Diseño: Estudio prospectivo de cohortes. Ámbito: Unidad de cuidados intensivos de un hospital terciario. Pacientes: Las primeras 22 muertes consecutivas por COVID-19. Intervenciones: Se llevaron a cabo biopsias pulmonares y análisis histopatológicos en pacientes fallecidos por SDRA asociado a COVID-19. Se evaluaron los datos clínicos y la evolución médica. Resultados: La mediana de edad de los pacientes fue de 66 (63-74) años y el 73% eran varones. La mediana de la duración de la ventilación mecánica fue de 17 (8-24) días. La lesión pulmonar inducida por COVID-19 se caracterizó por una fase exudativa durante la primera semana de la enfermedad, seguida de una fase proliferativa/organizativa en la segunda y tercera semana y, por último, una fase de fibrosis en fase terminal tras la tercera semana de evolución. Se detectaron proteínas y ARN vírico en neumocitos y macrófagos en una fase muy temprana de la enfermedad, pero estos ya no se volvieron a detectar a partir de la segunda semana. Limitación: Tamaño limitado de la muestra. Conclusión: La evolución cronológica de las lesiones histopatológicas pulmonares asociadas a la COVID-19 parece ser similar a la de otras formas de SDRA. En particular, se observan daños pulmonares coherentes con las lesiones potencialmente sensibles a los corticosteroides.