Balance between alpha-1Antitrypsin and Human Neutrophil Elastase (a1AT/HNE) in Bronchoalveolar Lavage fluid (BALf) of severe COVID19 patients

Neutrophils (Neu) play a pathogenic role in COVID19 by releasing Neutrophils Extracellular Traps (NETs) or HNE. Being HNE inhibited by a1AT, supplementation of this protein has been proposed. We aim to study a1AT/HNE balance in BALf from ICU admitted COVID19 patients. To assess HNE, a1AT and HNE/a1AT complexes, 33 COVID 19 BALf samples were analysed by means of ELISA or gel-Electrophoresis + Western Blot. Proteins bound to a1AT or HNE were identified by Liquid chromatography-mass spectrometry. NETs release (PMA stimulated Neu +/- a1AT) was analysed by confocal microscopy. Both HNE and a1AT were clearly detectable in BALf at high levels. Contrary to what previously observed in other settings (Bronchiolits obliterans) (Cagnone, M. et al. High Throughput 2019;8(1):5) we couldn't detect any HNE/ a1AT complex in COVID19 even when purified HNE was added to samples (Fig 1a). HNE was found to be bound to acute phase proteins, histones and C3. Due to the relevant role of NETs, we assessed the ability of free a1AT to bind to histones. Although this binding was confirmed, a1AT wasn't able to inhibit NETs formation (Fig 1b). Despite the finding of a high burden of free and bound HNE in COVID 19 BALf, the formation of HNE/ a1AT inhibitory complex is prevented. Furthermore, a1AT binds to histones but does not prevent NETs formation and their noxious activity.

Delayed immunological reaction of Human Microvascular Lung Endothelium in response to Human Coronavirus 229E infection

Introduction: In the natural conditions first and major target for respiratory viruses (RVs) are epithelial cells. Nonetheless, recently we have demonstrated that RVs are able to infect not only epithelium, but also Human Microvascular Lung Endothelial Cells (HMVEC-L) which increased network is observed during severe asthma due to increased angiogenesis. Furthermore, on the surface of HMVEC-L we observed intense expression of aminopeptidase N (AP-N)- an entry receptor for Human Coronavirus 229E (HCoV-229E). Due to the facts, that possibility of being infected by HCoV-229E should be considered and there is no research based on this model the aim of this study was to assess the vulnerability of HMVEC-L to HCoV-229E infection. Method(s): HMVEC-L was incubated with HCoV-229E (MOI 0,1;1,0;3,0) for 3 hours, 3x PBS washed and cultured for 120 hours. In relevant time points (5;24;48;72;96 and 120h) viral copy number and mRNA expression of inflammatory, anti-viral and receptor factors were evaluated in Real-Time PCR. Confocal microscopy (CM) and flow cytometry (FACS) were used to measure AP-N surface expression. Result(s): FACS and CM confirmed intense surface expression of AP-N on HMVEC-L. HCoV-229E efficiently infected HMVEC-L (604 945,5 +/-194 930,2 viral copies/mul) in 48h cultures (MOI 0,1) and induced relatively late (between 72- 96h) mRNA expression of RANTES (1181,12);IL-6 (89,6);IFN-beta (53,7);OAS-1 (64,3);PKR (11,4) and TLR-3 (42,4). Increased mRNA expression was also accompanied by protein release to the supernatants. Conclusion(s): HCoV-229E may efficiently infect HMVEC-L and induce delayed inflammatory and anti-viral response.

Recovery of Corneal Innervation after Treatment in Dry Eye Disease: A Confocal Microscopy Study.

PURPOSE: To analyze the changes in corneal innervation by means of in vivo corneal confocal microscopy (IVCM) in patients diagnosed with Evaporative (EDE) and Aqueous Deficient Dry Eye (ADDE) and treated with a standard treatment for Dry Eye Disease (DED) in combination with Plasma Rich in Growth Factors (PRGF). METHODS: Eighty-three patients diagnosed with DED were enrolled in this study and included in the EDE or ADDE subtype. The primary variables analyzed were the length, density and number of nerve branches, and the secondary variables were those related to the quantity and stability of the tear film and the subjective response of the patients measured with psychometric questionnaires. RESULTS: The combined treatment therapy with PRGF outperforms the standard treatment therapy in terms of subbasal nerve plexus regeneration, significantly increasing length, number of branches and nerve density, as well as significantly improving the stability of the tear film (p < 0.05 for all of them), and the most significant changes were located in the ADDE subtype. CONCLUSIONS: the corneal reinnervation process responds in a different way depending on the treatment prescribed and the subtype of dry eye disease. In vivo confocal microscopy is presented as a powerful technique in the diagnosis and management of neurosensory abnormalities in DED.

Update on Corneal Confocal Microscopy Imaging.

In vivo corneal confocal microscopy (IVCM) is a non-invasive ophthalmic imaging technique that provides images of the cornea at the cellular level. Despite the uses in ocular surface pathologies, in the last decades IVCM has been used to provide more knowledge in refractive surgery wound healing, in neuropathies diagnosis, etc. The observation of the corneal cells, both normal and inflammatory, and the possibility of quantification of the corneal nerve density with manual or automated tools, makes IVCM have a significant potential to improve the diagnosis and prognosis in several systemic and corneal conditions.

Identification of a red cell hemostatic mechanism causing COVID-19 microvascular obstruction

Background: Severe COVID-19 infection is associated with a wide spectrum of clinical manifestations, leading to systemic thromboinflammation and multiorgan dysfunction. The primary cause of multiorgan damage is widespread endothelial injury, leading to microangiopathy and organ ischemia. The molecular mechanisms by which ischemic endothelial cells causes microvascular obstruction remains ill defined. Aim(s): Identification of distinct microvascular occlusion mechanisms in COVID-19. Method(s): The microvasculature of multiple organs from patients dying from COVID-19, myocardial infarction or stroke were analyzed by H&E, immunohistochemistry, SEM and CLEM. Animal models of gut ischemia and stroke were also examined. Intravital confocal microscopy examined endothelial injury and microvascular obstruction mechanisms mediated by platelets, red cells and fibrin. Result(s): We demonstrate the existence of a distinct microvascular hemostatic mechanism mediated by hemolyzed red blood cells (RBC), independent of platelets and fibrin. Extensive RBC hemolysis was apparent in the microvasculature of COVID-19 patients and in humans with major organ ischemia, leading to widespread microvascular obstruction. This RBC hemostatic mechanism was triggered by organ ischemia and associated with localized accumulation of hemolyzed RBCs at sites of endothelial necroptosis. RBC hemolysis was impaired in animals lacking the necroptosis mediator, MLKL or the C9 component of complement, indicating the involvement of cell intrinsic and extrinsic membrane lytic processes. Intravital microscopy revealed that the RBC hemostatic mechanism was triggered by the fragmentation of lyzed RBCs and the deposition of RBC membranes on the surface of dying endothelial cells, forming an endovascular sealant that prevents interstitial bleeding. Conclusion(s): Our studies demonstrate the existence of a previously unidentified microvascular hemostatic mechanism mediated by hemolyzed RBCs. Dysregulation of this hemostatic mechanism is linked to microvascular obstruction and bleeding in COVID-19 and ischemic diseases.

COVID-19 disease is associated with a hypofibrinolytic state driven by elevated plasminogen activator inhibitor-1 and its cofactor vitronectin

Background: COVID-19 disease arises from infection with severe acute respiratory cornonavirus-2 (SARS-CoV- 2). Severe disease is associated with a coagulopathy characterised by elevated D-dimer levels, fibrin deposition in the lung, and a thrombotic incidence of approximately 30%, indicating catastrophic derailment of the haemostatic system. Aim(s): To investigate whether SARS-CoV- 2- induced coagulopathy arises due to an imbalance in the fibrinolysis. Method(s): Citrated plasma was collected from 139 patients presenting with symptomatic COVID-19, 24 patients with non-COVID- 19 respiratory infection and 30 healthy controls in a dual-centre study. Fibrinolytic biomarkers were evaluated including plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator (tPA), plasminogen, vitronectin and thrombin activatable fibrinolysis inhibitor (TAFI). Furthermore, diagnostic biomarkers including, fibrinogen, C-reactive protein (CRP), D-dimer and inflammatory cytokines were quantified. Clot lysis was evaluated using turbidity assays, plasma clot structure visualised by confocal microscopy and plasmin generation quantified by chromogenic substrate. Result(s): PAI-1 antigen, activity, and the cofactor for this serpin, vitronectin, were significantly elevated in patients with COVID-19 compared to healthy controls and non-COVID- 19 respiratory infection. Patients with COVID-19 exhibit attenuated plasmin generation compared to healthy volunteers despite significant elevation in tPA. PAI-1 correlated with inflammatory cytokines (IL-1beta, IL-8 and TNF-alpha). In line with this acute phase proteins, fibrinogen and CRP were high in patients with COVID-19 but only CRP was increased compared to non-COVID- 19 respiratory infections. Levels of PAI-1 and vitronectin were associated with escalating oxygen support and a corresponding decrease in plasminogen. Importantly, patients with COVID-19 disease exhibit resistance to fibrinolytic degradation by Actilyse, however, this could be overcome by the PAI-1 resistant form of tPA, Metalyse. Conclusion(s): We reveal that COVID-19 disease promotes a hypofibrinolytic state due to elevated PAI-1 and its stabilizing cofactor vitronectin. PAI-1 correlates with inflammatory cytokines and disease severity thereby highlighting its potential prognostic power in the development of severe COVID-19 disease.

Altered fibrin network structure and fibrinolysis in intensive care unit patients with COVID-19, not entirely explaining the increased risk of thrombosis

Background: SARS-CoV- 2 infection is associated with an increased incidence of thrombosis. Aim(s): By studying the fibrin network structure of COVID-19 patients, we aimed to unravel pathophysiological mechanisms that contribute to this increased risk of thrombosis. This may contribute to optimal prevention and treatment of COVID-19 related thrombosis. Method(s): In this case-control study, we collected plasma samples from intensive care unit (ICU) patients with COVID-19, with and without confirmed thrombosis, between April and December 2020. Additionally, we collected plasma from COVID-19 patients admitted to general wards without thrombosis, from ICU patients with pneumococcal infection, and from healthy controls. Fibrin fiber diameters and fibrin network density were quantified in plasma clots imaged with stimulated emission depletion (STED) microscopy and confocal microscopy. Finally, we determined the sensitivity to fibrinolysis. Ethical approval was obtained and written informed consent was obtained or an opt-out procedure was in place. Result(s): COVID-19 ICU patients (n = 37) and ICU patients with pneumococcal disease (n = 7) showed significantly higher fibrin network densities and longer plasma clot lysis times than healthy controls (n = 7) (Figure 1). No differences were observed between COVID-19 ICU patients with and without thrombosis, or ICU patients with pneumococcal infection. At a second time point, after thrombosis or at a similar time point in patients without thrombosis, we observed thicker fibers and longer lysis times in COVID-19 ICU patients with thrombosis (n = 19) than in COVID-19 ICU patients without thrombosis (n = 18). Conclusion(s): Our results suggest that severe COVID-19 is associated with a changed fibrin network structure and decreased susceptibility to fibrinolysis. Since these changes were not exclusive to COVID-19 patients, they may not explain the increased thrombosis risk. (Figure Presented).

Inversed platelet/fibrin ratio in shear-dependent thrombus formation between patients with COVID-19 and sepsis identifies subtherapeutic GPIIb/IIIa blockade as a potential therapeutic target

Background: Thrombotic events are frequent and life-threating complications of COVID-19, but are also observed in patients with bacterial sepsis. Disseminated thrombosis may occur despite strict anticoagulation, suggesting that platelets play a direct, but yet undefined role. Several studies demonstrate altered platelet function in COVID-19, but the impact of platelets in COVID-19 and sepsis remains poorly understood. Aim(s): Platelet phenotype and function were comprehensively assessed in over 100 patients with either COVID-19 (non-ECMO, all-ICU, n = 23), bacterial infection without sepsis (SOFA-score < 2;n = 29), or sepsis/septic shock (SOFA-score >=2;n = 49) at multiple time points during the disease. Method(s): Patients were recruited at the local University Hospital (Ethical vote 94/19). Platelet phenotype and function were studied using flow cytometry (lumino-) aggregometry and whole-mount transmission electron-microscopy. Thrombus formation was investigated using a collagen-and tissue factor-coated flow chamber model at arterial shear rate (1000s-1). Thrombi were imaged by confocal microscopy. Result(s): Upon stimulation with ADP or CRP-XL platelets of infection patients without sepsis showed reduced PAC-1 binding and CD62P exposition. In sepsis patients reactivity was even more impaired and highly associated with disease severity (mean normalized geo-MFI PAC-1 infection: 0.56 vs sepsis: 0.25, p < 0.01;ROC-AUC: 0.76, p < 0.001). Intriguingly, platelets of COVID-19 patients were more responsive towards stimulation compared to comparably-ill ICU patients with sepsis. This relative hyper-reactivity was reflected by increased clot-formation in the flow chamber, compared to sepsis patients (mean surface coverage: 36% vs. 19%, p < 0.05). Thrombi of COVID-19 patients were platelet-rich with little fibrin, in contrast to healthy donors or sepsis patients showing increased amount of fibrin and less platelets. Subtherapeutic doses of GPIIb/IIIa blockers eptifibatide or tirofiban, which had minor effect in control blood, sufficiently prevented thrombus formation in COVID-19 samples under arterial flow. Conclusion(s): Our findings provide evidence that low dose GPIIb/ IIIa blockade might act as a powerful therapeutic tool in COVID-19 patients.

Generation Of Macro-scale And Mature Lung Organoids Through Engineering Cell Microenvironment

Purpose/Objectives: <Most used lower respiratory tract models consist of cell monolayers which lack of tissue and organ level response and of in-vivo phenotype. Ex-vivo lung tissues have short viability and limited availability. Lung organoids, which recapitulates better the 3D cellular complex structures, architecture, and in-vivo function, fail to reach maturity even after 85 -185 days of culture. Therefore, these models have a limited use to study fetal lung diseases. Other lung models, consist of only one structure of the lower track, such as bronchial tubes or alveoli, but fail to recapitulate the whole organ structure. In this work, cell microenvironment was used to promote the self-organization of epithelial and mesenchymal cells into macro-structures, aiming to mimic the whole and adult lower respiratory tract model> Methodology: <Different parts of the microenvironment were considered to create a compliant matrix. Alginate-Gelatin hydrogels were used for 3D encapsulation of mesenchymal origin cells. This hydrogel provided a stiffness like the one on the lung. Base membrane zone proteins were used to induce the attachment and guidance of epithelial cells into 3D structures. The interactions between both cell types, further guided them into lung fate. The morphology of resulting organoids was analyzed using immunostaining and confocal microscopy, LSM710, with the purpose of evaluate polarization, protein markers, and different cell populations. Quantitative PCR was performed to evaluate and compare the expression of lung fate genes with traditional cell monocultures.> Results: <The engineered microenvironment and protocol development done in this work resulted in macro-scale structures, in which branching morphogenesis occurred at day 21. Different structures were identified in the organoid including bronchial tube, bronchioles, and alveoli. Polarization of the organoids was confirmed by visualization of E-cadherin, and ZO-1. Expression of Surfactant Protein B and C into the organoids confirmed the presence of alveolar type II cells, which are only present in the later development stage. Surfactant Protein B, Transmembrane protease, serine 2, TMPRSS-2, and Angiotensin-converting enzyme 2, ACE2 were found to be significantly higher expressed into the organoids in comparison with traditional epithelial cells monolayers.> Conclusion/Significance: <Growth factors are normally used to induce the fate of stem cells into lung organoids;however, these fail to reach maturity. Here, we developed a new methodology to induce the formation of the organoids based on the cell microenvironment. The resulting organoids require less time for development. The initial stage of adult cells can be modulated through culture conditions induce a 3D structure like the adult lung. As such, these organoids have the potential to be used for modeling adult diseases and to develop specific models from patient cells, which is one step forward to personalized medicine. SFTPB is one of the main proteins which facilitates the breathing process. Its high expression into our model may indicate that breathing occurs into our lung organoids. The higher expression of TMPRSS-2 and ACE2 into the organoids has a major significance in the field of virology since both proteins are the mainly entrance of SARS-CoV-2, and influenza H1N1.>.

Corneal innervation changes after Sars-Cov-2 Infection. An In vivo Confocal Microscopy Study

Purpose : To measure the innervation of the corneal subbasal nerve plexus of Covid-19 patients and compare the results with values of healthy patients. Methods : A prospective, observational study was conducted analyzing 39 eyes of patients who had overcome Covid -19 and 46 eyes of healthy volunteers included as a control group (verified by antibody analysis and negative qPCR result) which underwent in vivo confocal microscopy with Rodstock Cornea Module© attached to Heildelberg HRT3© . Ocular surgery procedures, previous ocular infections or systemic diseases that could cause alteration in corneal innervation were exclusion criteria. At least 5 non overlapping images of each eye were selected and only one eye of each patient was included in the study. Following sub basal nervous plexus parameters were measured with ACC Metrics © software: Corneal Nerve Fiber Density (CNFD), Corneal Nerve Branch Density (CNBD), Corneal Nerve Fiber Length (CNFL), Corneal Total Branch Density (CTBD), Corneal Nerve Fiber Area (CNFA), Corneal Nerve Fractal Dimension (CNFrD). Data analysis was performed with SPSS® software for Windows 22.0 (SPSS® Inc, Chicago, IL.). The differences of age and sex between groups were checked with T-test and chi-square tests. The normality of the sample was checked with the Shapiro-Wilk test and the results were compared with the T test or the Man-Whitney U test based on the distribution of the data. The differences were considered statistically significant for p<0.05. Results : There was no difference in the sex distribution between the groups (p= 0.248). The average age (± standard error) was 46.61±17.55 years for Covid-19 patients and 43.11±16.95 years for healthy control group (p=0.353) The mean of the analyzed variables (± standard error) from Covid-19 patients versus control group were CNFD: 16.09±6.92 and 23.03±8.31 fibers/mm2 (p=0,00008), CNBD: 21.93±15.37 and 28.93±17.84 branches/mm2 (p=0.064), CNFL: 11.61±3.61 and 14.05±3.71 mm/mm2 (p=0.002), CTBD: 38.48±20.02 and 43.29 ± 23.94 (p=0.41), CNFA: 0.0057±0.0017 and 0.006±0.0023 mm2 /mm2 (p=0.853), CNFrD: 1.46±0.041 and 1.47±0.037 (p=0.007). Conclusions : According to the data obtained, corneal subbasal nerve plexus is decreased in Covid-19 patients compared to the healthy control group, statistically significant for density, length, and fractal dimension. The results show the presence of possible small fiber neuropathy induced by Covid-19 disease.

Retinas of human donors with COVID-19 disease show morphological alterations and signs of inflammation including glial activation

Purpose : Different signs of inflammation have been described in the brains of COVID-19 patients. In the retina, the fundus eye exam of these patients shows cotton wool spots, microhemorrhages, and a decrease in vascular density. However, morphological alterations of retinal cells in these patients are unknown. Thus, the aim was to analyze the morphological changes of the retinal cells from human donors with COVID-19 to establish several stages of response to damage in these cells and to define correlations with clinical parameters. Methods : The retinas of human donors with COVID-19 (n = 16) and control subjects (n = 12) obtained from the General University Hospital Consortium of Valencia were analyzed. Immunohistochemical stainings were performed on transversal sections or flat-mount retinas to study photoreceptors, microglial cells, Müller cells, astrocytes, and the presence of ACE2. TUNEL assays and confocal microscopy imaging were carried out. Correlations were calculated between retinal and clinical parameters. Results : Mean age of COVID-19 and control group were 80±10 and 70±8 years respectively. Müller cells, outer segment of cones and retinal pigment epithelium presented ACE2 staining. Larger staining of ACE2 and CRALBP was observed in cell bodies of Müller cells in COVID group. Disorganization of honeycomb-like pattern formed by Müller cells in the outer nuclear layer and disruption of external limiting membrane was found in the 81.3% of COVID patients. The 56.3% of COVID patients showed gliosis compared to controls (40%). COVID-19 retinas also presented epiretinal membranes and astrocytes protruding to vitreous humor. The 93.8% of COVID-19 patients had activated or ameboid-shape microglia. Microglial nodules around vessels and a reduction of the area occupied by microglia in these retinas were observed. COVID-19 group showed a more severe degeneration of cones. Cone degeneration correlated with Müller cell activation. Age of COVID patients correlated inversely with total retinal degeneration. Conclusions : Morphological alterations in the cone photoreceptors as well as glial activation showing an inflammatory state of the retina were observed in COVID-19 patients.

Human interaction targets of SARS-COV-2 spike protein: A systematic review

Objectives: The development of effective targeted therapy and drug-design approaches against the SARS-CoV-2 is a universal health priority. Therefore, it is important to assess possible therapeutic strategies against SARS-CoV-2 via its most interaction targets. The present study aimed to perform a systematic review on clinical and experimental investigations regarding SARS-COV-2 interaction targets for human cell entry. Methods: A systematic search using relevant MeSH terms and keywords was performed in PubMed, Scopus, Embase, and Web of Science (ISI) databases up to July 2021. Two reviewers independently assessed the eligibility of the studies, extracted the data, and evaluated the methodological quality of the included studies. Additionally, a narrative synthesis was done as a qualitative method for data gathering and synthesis of each outcome measure. Results: A total of 5610 studies were identified, and 128 articles were included in the systematic review. Based on the results, spike antigen was the only interaction protein from SARS-CoV-2. However, the interaction proteins from humans varied including different spike receptors and several cleavage enzymes. The most common interactions of the spike protein of SARS-CoV-2 for cell entry were ACE2 (entry receptor) and TMPRSS2 (for spike priming). A lot of published studies have mainly focused on the ACE2 receptor followed by the TMPRSS family and furin. Based on the results, ACE2 polymorphisms as well as spike RBD mutations affected the SARS-CoV-2 binding affinity. Conclusion: The included studies shed more light on SARS-CoV-2 cellular entry mechanisms and detailed interactions, which could enhance the understanding of SARS-CoV-2 pathogenesis and the development of new and comprehensive therapeutic approaches.

IRE1A/ XBP1s Axis of ER Stress Promotes DRP1 Phosphorylation at Specific Serine Residue (S616) and Induces Mitochondrial Fragmentation During Airway Inflammation

RATIONALE: Airway inflammation plays a role in airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, and COVID-19 that affect millions of people worldwide. Previously, we showed that acute (24-h) exposure to the pro-inflammatory cytokine tumor necrosis factor α (TNFα) triggers an endoplasmic reticulum (ER) stress response in human airway smooth muscle (hASM) cells. In hASM cells, TNFα selectively activates the inositol requiring enzyme 1α (IRE1α) ER stress pathway with downstream splicing of X-box binding protein 1 (XBP1s), which transcriptionally activates expression of target genes that include proteins mediating phosphorylation of dynamin-related protein 1 (pDRP1) at the Ser616 (S616) residue. Increased pDRP1 at S616 is associated with mitochondrial fission (fragmentation);however, DRP1 is also phosphorylated at Ser637 (S637) residue, and the balance between phosphorylation at S616 and S637 regulates the translocation of DRP1 from cytosol to mitochondria and subsequent fragmentation of mitochondria. In the present study, we hypothesized that TNFα induces ER stress leading to XBP1s mediated increase in the expression of specific kinases that phosphorylate DRP1 at S616 and promote mitochondrial fragmentation. METHODS: hASM cells, dissociated from bronchial tissue obtained from patients with no history of respiratory diseases, were exposed to TNFα (20 ng/ ml for 6-h). As an inhibitor of fragmentation, cells were treated with Mdivi1 (50 μM for 6-h), GTPase inhibitor of DRP1. The expression and phosphorylation status of IRE1α, DRP1, XBP1, cyclin dependent kinases (CDK1, CDK5) and cyclin B1 were quantified by Western blot and immunohistochemistry. Mitochondrial morphology was assessed by 3D confocal microscopy using MitoTracker. XBP1-targets were confirmed by chromatin immunoprecipitation (ChIP) and quantitative PCR. RESULTS: Bioinformatics analysis predicted putative binding sites of XBP1 in the promoter region of CDK1, CDK5 and cyclin B1 genes that are reported to phosphorylate DRP1 at S616. Consistent with our previous findings, we found that TNFα increases IRE1α phosphorylation and XBP1 splicing. The TNFα induced increase in XBP1s transcriptionally activated expression of CDK1, CDK5 and cyclin B1 and leads to subsequent phosphorylation of DRP1 at S616 with no change in S637 phosphorylation. As a result, TNFα mediated increase in the ratio of S616/ S637 phosphorylation, which promoted translocation of DRP1 from cytosol to mitochondria and mitochondrial fragmentation. We also showed that Mdivi1 mediated inhibition of DRP1-GTPase activity ameliorated phosphorylation at S616 residue and significantly reduced mitochondrial fragmentation. CONCLUSIONS: The present study elucidates the mechanism underlying TNFα induced ER stress and mitochondrial fragmentation.

Plasma Neutrophil Extracellular Trap Levels Correlate with Acute Respiratory Distress Syndrome Disease Severity

Rationale: Acute Respiratory Distress Syndrome (ARDS) is characterized by acute onset of hypoxic respiratory failure, which can be complicated by multi-organ dysfunction and death. ARDS results from inflammatory alveolar injury precipitated by direct or indirect lung injury. Neutrophils play a central role in the pathology of ARDS and release neutrophil extracellular traps (NETs) to trap and kill pathogens. Dysregulated NET formation, however, can cause inflammatory tissue damage and exacerbate acute lung injury as in COVID-19 associated ARDS. Whether detection of plasma NETs predicts outcomes in non-COVID-19 associated ARDs remains unknown. We hypothesized that plasma NET levels correlate directly with disease severity and mortality in non-COVID-19 ARDS patients. Methods: We obtained previously collected plasma samples from patients (n=200) with moderate to severe ARDS enrolled in the Re-evaluation of Systemic Early Neuromuscular Blockade (ROSE) trial at three different time points (admission, 24 hours, and 48 hours after admission) complete with clinical outcome data through 28 days after admission. We also examined age- and gender-matched healthy donor plasma (n=20). We assayed cell-free DNA levels via fluorescence and MPO-DNA complexes as a surrogate for NETs in each plasma sample. Clinical outcomes from ROSE trial participants were correlated with the quantification of NETs. We also assessed NET formation by neutrophils isolated from healthy adults following incubation with ARDS patient and healthy donor plasma samples using live cell imaging and confocal microscopy. Results: We demonstrated elevated plasma markers of NETs (cell-free DNA and MPO-DNA complexes) in ARDS plasma compared to healthy donor plasma. Deceased study participants demonstrated higher plasma NET levels on admission and at 48 hours as compared to ARDS survivors (admission: p = 0.0045 and 48 hours: p = 0.0050). Increased plasma NETs on admission, at 24 hours, and 48 hours also correlated with illness severity. Furthermore, ARDS plasma samples induced NET formation in vitro in neutrophils isolated from healthy donors while control plasma did not. Conclusion: NET formation is increased in plasma from patients with ARDS compared to healthy donor plasma, consistent with the inflammatory alveolar injury seen in ARDS. Additionally, plasma from ARDS patients induces NET formation in vitro in PMNs isolated from healthy adult donors. We speculate that exaggerated NET formation may serve as a novel biomarker for inflammatory lung injury in ARDS resulting from multiple etiologies and that strategies targeting NET formation may improve outcomes in ARDS.

Neuropathic Corneal Pain as Debilitating Manifestation of LONG-COVID.

This report illustrates the case of a female patient suffering from severe ocular discomfort, tinnitus and ageusia, 7 months after a SARS-CoV-2 infection. The medical history implicated a diagnosis of LONG-COVID with ocular pain as the most debilitating symptom. In-vivo confocal microscopy revealed corneal microneuromas with hyperreflectivity and irregular enlargement of nerve endings in both eyes, which led to the diagnosis of neuropathic corneal pain. The aim of this report is to increase awareness that COVID-19 induced neuropathic pain can also occur in the cornea representing the human body's most richly innervated tissue.

EFFECT of ASTODRIMER SODIUM AGAINST SARS-CoV-2 VARIANTS (Α, Β, Γ, Δ, Κ) in VITRO

Background: The dominance of SARS-CoV-2 Variants of Concern (VOC) and Interest (VOI) has challenged the efficacy of public health strategies to control the current pandemic. Astodrimer sodium is a broad-spectrum antiviral dendrimer that has been formulated as a topical nasal spray to help reduce exposure to infectious viral load in the nasal cavity. Astodrimer sodium showed antiviral and virucidal activity against early pandemic isolates of SARS-CoV-2 in vitro and after nasal administration in vivo. The current studies assessed the spectrum of activity of astodrimer sodium against emerging variants of SARS-CoV-2 and other pandemic viruses. Methods: Assays utilized hACE2+ and hTMPRSS2+ HEK-293T cells, Calu-3 and Vero E6 cells. Time of addition studies involved adding astodrimer sodium 1 hour prior to, at the time of, or 1-hour post-infection. Coronavirus spike receptor binding domain (RBD) or S1 binding studies were analysed by ELISA or confocal microscopy. Virucidal studies involved exposing 105 SARS-CoV-2 PFU to 10mg/mL astodrimer sodium for 0.5, 1, 5, 15 and 30 mins. Results: Astodrimer sodium demonstrated potent antiviral and virucidal activity against SARS-CoV-2 VOC α, β, δ and γ, and VOI κ in Vero E6 and Calu-3 cells. Astodrimer sodium reduced infectious viral load of all variants by >99.9% vs virus control. The pan-SARS-CoV-2 activity of astodrimer sodium occurred despite multiple mutations and deletions in the viral spike protein of each variant. The attachment of SARS-CoV-2 early pandemic virus isolates, Wuhan-Hu-1 and USA-WA-1/2020, and SARS-CoV-1 Spike binding to ACE2, as well as attachment of Middle Eastern respiratory syndrome (MERS) coronavirus spike protein to its cellular receptor, was inhibited by astodrimer sodium. Astodrimer sodium did not prevent attachment of the SARS-CoV-2 VOC α and β spike S1, or γ RBD spike protein, to the ACE2 receptor in vitro. Conclusion: Astodrimer sodium mimics negatively charged glycosaminoglycans and provides a potent antiviral and virucidal barrier to viral attachment and entry. The potent broad-spectrum anti-pandemic coronavirus and virucidal efficacy of astodrimer sodium against whole virus is likely due to blocking multiple electrostatic interactions of the spike protein that are not negated by minor or major changes to the isolated RBD of SARS-CoV-2 VOC α, β and γ alone. Astodrimer sodium has the potential to block the binding of pan-SARS-CoV-2, thus reducing the potential for the development of COVID-19.

Animal Models of COVID-19: Nonhuman Primates.

With the advent of the novel SARS-CoV-2, the entire world has been thrown into chaos with severe disruptions from a normal life. While the entire world was going chaotic, the researchers throughout the world were struggling to contribute to the best of their capabilities to advance the understanding of this new pandemic and fast track the development of novel therapeutics and vaccines. While various animal models have helped a lot to understand the basic physiology, nonhman primates have been promising and much more successful in modelling human diseases compared to other available clinical models. Here we describe the different aspects of modelling the SARS-CoV-2 infection in NHPs along with the associated methods used in NHP immunology.

Herpes Simplex Virus 1 Anterior Uveitis following Coronavirus Disease 2019 (COVID-19) Vaccination in an Asian Indian Female.

PURPOSE: To describe a case of herpes simplex virus 1 (HSV 1) infection following coronavirus disease 2019 (COVID-19) vaccination in an Asian Indian female. METHODS: Retrospective case report. RESULT: A 40-year-old female presented with decreased vision, pain, and photophobia of 2 weeks duration. She reported receiving the second dose of COVISHIELDTM (ChAdOx1-S [recombinant]) 1 week prior to the onset of ocular symptoms. Left eye examination revealed granulomatous anterior uveitis. Aqueous sample from the left eye tested positive for HSV1 by polymerase chain reaction(PCR) method. She was managed with oral antiviral therapy, topical steroids, and cycloplegic agent and showed significant improvement of inflammation within 1 week and resolved within 3 weeks. CONCLUSION: This report demonstrates a potential association of HSV 1 anterior uveitis with COVID-19 vaccination. A high index of suspicion of viral etiology is warranted when uveitis presents with reduced corneal sensations and pigmented keratic precipitates, following a recent history of COVID-19 vaccination.

Small Fibre Peripheral Alterations Following COVID-19 Detected by Corneal Confocal Microscopy.

A large spectrum of neurological manifestations has been associated with coronavirus disease 2019 (COVID-19), and recently, the involvement of small fibers has been suggested. This study aims to investigate the involvement of small peripheral nervous fibers in recovered COVID-19 patients using in-vivo corneal confocal microscopy (CCM). Patients recovered from COVID-19 and a control group of healthy subjects underwent in-vivo CCM. Corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), corneal nerve fiber length (CNFL), corneal nerve fiber total branch density (CTBD), corneal nerve fiber area (CNFA), corneal nerve fiber width (CNFW), fiber tortuosity (FT), number of beadings (NBe), and dendritic cells (DC) density were quantified. We enrolled 302 eyes of 151 patients. CNBD and FT were significantly higher (p = 0.0131, p < 0.0001), whereas CNFW and NBe were significantly lower (p = 0.0056, p = 0.0045) in the COVID-19 group compared to controls. Only CNBD and FT resulted significantly correlated to antiviral drugs (increased) and corticosteroids (decreased). No significant relationship with disease severity parameters was found. COVID-19 may induce peripheral neuropathy in small fibers even months after recovery, regardless of systemic conditions and therapy, and CCM may be a useful tool to identify and monitor these morphological changes.