Exploration of potential mechanism of SARS-CoV-2 induced immune inflammatory response leading to progression of atherosclerosis and prediction of traditional Chinese medicine for preventing and treating based on bioinformatics.

Objective To explore the core targets and important pathways of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) induced atherosclerosis (AS) progression from the perspective of immune inflammation, so as to predict the potential prevention and treatment of traditional Chinese medicine (TCM). Methods Microarray data were obtained from the Gene Expression Omnibus (GEO) database for coronavirus disease 2019 (COVID-19) patients and AS patients, and the "limmar" and "Venn" packages were used to screen out the common differentially expressed genes (DEGs) genes in both diseases. The gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses were performed on the common DEGs to annotate their functions and important pathways. The two gene sets were scored for immune cells and immune function to assess the level of immune cell infiltration. The protein-protein interaction (PPI) network was constructed by STRING database, and the CytoHubba plug-in of Cytoscape was used to identify the hub genes. Two external validation datasets were introduced to validate the hub genes and obtain the core genes. Immuno-infiltration analysis and gene set enrichment analysis (GSEA) were performed on the core genes respectively. Finally the potential TCM regulating the core genes were predicted by Coremine Medical database. Results A total of 7898 genes related to COVID-19, 471 genes related to AS progression;And 51 common DEGs, including 32 highly expressed genes and 19 low expressed genes were obtained. GO and KEGG analysis showed that common DEGs, which were mainly localized in cypermethrin-encapsulated vesicles, platelet alpha particles, phagocytic vesicle membranes and vesicles, were involved in many biological processes such as myeloid differentiation factor 88 (MyD88)-dependent Toll-like receptor signaling pathway transduction, interleukin-8 (IL-8) production and positive regulation, IL-6 production and positive regulation to play a role in regulating nicotinamide adenine dinucleotide phosphate oxidase activity, Toll-like receptor binding and lipopeptide and glycosaminoglycan binding through many biological pathways, including Toll-like receptor signaling pathways, neutrophil extracellular trap formation, complement and coagulation cascade reactions. The results of immune infiltration analysis demonstrated the state of immune microenvironment of COVID-19 and AS. A total of 5 hub genes were obtained after screening, among which Toll-like receptor 2 (TLR2), cluster of differentiation 163 (CD163) and complement C1q subcomponent subunit B (C1QB) genes passed external validation as core genes. The core genes showed strong correlation with immune process and inflammatory response in both immune infiltration analysis and GSEA enrichment analysis. A total of 35 TCMs, including Chuanxiong (Chuanxiong Rhizoma), Taoren (Persicae Semen), Danggui (Angelicae Sinensis Radix), Huangqin (Scutellariae Radix), Pugongying (Taraxaci Herba), Taizishen (Pseudostellariae Radix), Huangjing (Polygonati Rhizoma), could be used as potential therapeutic agents. Conclusion TLR2, CD163 and C1QB were the core molecules of SARS-CoV-2-mediated immune inflammatory response promoting AS progression, and targeting predicted herbs were potential drugs to slow down AS progression in COVID-19 patients.Copyright © 2023 Editorial Office of Chinese Traditional and Herbal Drugs. All rights reserved.

Therapeutic approaches for intravascular microthrombi-induced acute respiratory distress syndrome (ARDS) in COVID-19 infection.

The COVID-19 pandemic has overwhelmed our health care capacity in an unprecedented way due to the sheer number of critically infected patients admitted to hospitals during the last two years. Endothelial injury is seen as one of the central hallmarks of COVID-19 infection that is the starting point in the generation of microthrombi and sepsis eventually leading to acute respiratory distress syndrome (ARDs) and multi-organ failure. The dramatic fall in lung function during ARDs is attributed to the microthrombi-induced coagulopathy primed by a hyperactive immune system. Due to the lack of effective antiviral agents, the line of treatment is limited to the management of two key risk factors i.e., immune activation and coagulopathy. In the present review, we describe the mechanistic role, therapeutic targets and opportunities to control immune activation and coagulopathy during the pathogenesis of COVID-19-induced ARDs.

Association of complement pathways with COVID-19 severity and outcomes.

OBJECTIVES: Complement activation has been implicated in COVID-19 pathogenesis. This study aimed to assess the levels of complement activation products and full-length proteins in hospitalized patients with COVID-19, and evaluated whether complement pathway markers are associated with outcomes. METHODS: Longitudinal measurements of complement biomarkers from 89 hospitalized adult patients, grouped by baseline disease severity, enrolled in an adaptive, phase 2/3, randomized, double-blind, placebo-controlled trial and treated with intravenous sarilumab (200 mg or 400 mg) or placebo (NCT04315298), were performed. These measurements were then correlated with clinical and laboratory parameters. RESULTS: All complement pathways were activated in hospitalized patients with COVID-19. Alternative pathway activation was predominant earlier in the disease course. Complement biomarkers correlated with multiple variables of multi-organ dysfunction and inflammatory injury. High plasma sC5b-9, C3a, factor Bb levels, and low mannan-binding lectin levels were associated with increased mortality. Sarilumab treatment showed a modest inhibitory effect on complement activation. Moreover, sera from patients spontaneously deposited C5b-9 complex on the endothelial surface ex vivo, suggesting a microvascular thrombotic potential. CONCLUSION: These results advance our understanding of COVID-19 disease pathophysiology and demonstrate the importance of specific complement pathway components as prognostic biomarkers in COVID-19.

Complement and complement regulatory proteins are upregulated in lungs of COVID-19 patients.

We explored the pathological changes and the activation of local complement system in COVID-19 pneumonia. Lung paraffin sections of COVID-19 infected patients were analyzed by HE (hematoxylin-eosin) staining. The deposition of complement C3, the deposition of C3b/iC3b/C3d and C5b-9, and the expression of complement regulatory proteins, CD59, CD46 and CD55 were detected by immunohistochemistry. In COVID-19 patients' lung tissues, fibrin exudation, mixed with erythrocyte, alveolar macrophage and shed pneumocyte are usually observed in the alveoli. The formation of an "alveolar emboli" structure may contribute to thrombosis and consolidation in lung tissue. In addition, we also found that compared to normal tissue, the lung tissues of COVID-19 patients displayed the hyper-activation of complement that is represented by extensive deposition of C3, C3b/iC3b/C3d and C5b-9, and the increased expression level of complement regulatory proteins CD55, and especially CD59 but not CD46. The thrombosis and consolidation in lung tissues may contribute to the pathogenesis of COVID-19. The increased expression of CD55 and CD59 may reflect a feedback of self-protection on the complement hyper-activation. Further, the increased C3 deposition and the strongly activated complement system in lung tissues may suggest the rationale of complement-targeted therapeutics in conquering COVID-19.

Neurologic sequelae of COVID-19 are determined by immunologic imprinting from previous coronaviruses.

Coronavirus disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global public health emergency. Although SARS-CoV-2 is primarily a respiratory pathogen, extra-respiratory organs, including the central nervous system (CNS), can also be affected. Neurologic symptoms have been observed not only during acute SARS-CoV-2 infection, but also at distance from respiratory disease, also known as long-COVID or neurological post-acute sequelae of COVID-19 (neuroPASC). The pathogenesis of neuroPASC is not well understood, but hypotheses include SARS-CoV-2-induced immune dysfunctions, hormonal dysregulations, and persistence of SARS-CoV-2 reservoirs. In this prospective cohort study, we used a high throughput systems serology approach to dissect the humoral response to SARS-CoV-2 (and other common Coronaviruses - 229E, HKU1, NL63, OC43) in the serum and cerebrospinal fluid (CSF) from 112 infected individuals who developed (n = 18) or did not develop (n = 94) neuroPASC. Unique SARS-CoV-2 humoral profiles were observed in the CSF of neuroPASC, compared to serum responses. All antibody isotypes (IgG, IgM, IgA) and subclasses (IgA1-2; IgG1-4) were detected in serum, whereas CSF was characterized by focused IgG1 (and absence of IgM). These data argue in favor of compartmentalized brain-specific responses against SARS-CoV-2 through selective transfer of antibodies from the serum to the CSF across the blood-brain-barrier, rather than intrathecal synthesis, where more diversity in antibody classes/subclasses would be expected. Compared to individuals who did not develop post-acute complications following infection, individuals with neuroPASC had similar demographic features (median age 65 vs 66.5 years, respectively, p = 0.55; females 33% vs 44%, p = 0.52), but exhibited attenuated systemic antibody responses against SARS-CoV-2, characterized by decreased capacity to activate antibody-dependent complement deposition (ADCD), NK cell activation (ADNKA) and to bind Fcγ receptors. However, surprisingly, neuroPASC individuals showed significantly expanded antibody responses to other common Coronaviruses, including 229E, HKU1, NL63, and OC43. This biased humoral activation across coronaviruses was particularly enriched in neuroPASC individuals with poor outcome, suggesting an original antigenic sin (or immunologic imprinting), where pre-existing immune responses against related viruses shape the response to current infection, as a key prognostic marker of neuroPASC disease. Overall, these findings point to a pathogenic role for compromised anti-SARS-CoV-2 responses in the CSF, likely resulting in incomplete virus clearance from the brain and persistent neuroinflammation, in the development of post-acute neurologic complications of SARS-CoV-2 infection.

COVID-19 and kidneys: A follow-up.

In coronavirus disease 2019 (COVID-19) patients, acute tubular injury is the most frequently documented kidney disease. According to the current knowledge, its cause is assumed to be multifactorial. Other kidney diseases observed in non-severely ill COVID-19 patients are thrombotic microangiopathy, necrotizing glomerulonephritis, primary podocytopathy and interstitial nephritis. Even after a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination, necrotizing glomerulonephritis and other kidney diseases were observed. It is recommended that a renal biopsy be performed in COVID-19 patients with elevated creatinine, proteinuria, and/or hematuria to rule out a variety of other renal disorders. Both diseases (during a SARS-CoV-2 infection and after vaccination) probably share common features that act as triggers when the patient is preconditioned for a renal disease. The activation of the complement system and the formation of neutrophil extracellular traps (NET) could play a role in the pathogenesis. As the first report on autopsies carried out on COVID-19 patients throughout Germany showed, the autopsy plays a central role for a better understanding of this (relatively) new disease.Copyright © 2022, The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.

Peculiarities of Periodontal Pocket Microbiome in Patients with Generalized Periodontitis in the Post-Covid Period

The oral cavity, like the lungs, is often referred to as the <<ecological niche of commensal, symbiotic, and pathogenic or-ganisms,>> and the emigration and elimination of microbes between them are constant, ensuring a healthy distribution of saprophytic microorganisms that maintains organ, tissue, and immune homeostasis. The prolonged hospital stays due to COVID-19 complications, cross-infection, oxygenation therapy through the mask or incubation, and long-term intravenous infusions limit the patient's ability to care about the oral cavity, regularly clean teeth, floss interdental, etc., which creates extremely favorable conditions for colonization by aerobic and anaerobic pathogens of the oral cavity and periodontal pockets and leads to the rapid progression of chronic generalized periodontitis in this category of patients in the future. The goal of the study was to assess the state of the microbiome of the periodontal pockets of dental patients in the post-covid period. Methods. The object of the study was 140 patients with generalized periodontitis of the I and II stages of development in the chronic course (GP), among which 80 patients had coronavirus disease in the closest past. The patients were randomized by age, sex, and stage of GP development. The diagnosis of periodontal disease was established according to the classification by Danilevskyi. The bacteriological material for aerobic and facultative anaerobic microflora and yeast-like fungi was collected from periodontal pockets with a calibrated bacteriological loop and immediately seeded on blood agar. Results. Significant qualitative and quantitative changes in the nature of the oral microbiocenosis were observed in patients with GP after the recent coronavirus disease, compared with similar patients who did not suffer from COVID-19. We have noticed almost complete disappearance of bacteria that belong to the transient representatives of the oral microflora such as Neisseria, corynebacteria (diphtheria), micrococci, and lac-tobacilli. The main resident representatives of the oral microflora, i.e., alpha-hemolytic Streptococci of the mitis group, were found in all healthy individuals and patients of groups A and C, but in 30.0 +/- 4.58% of patients in group B, alpha-hemolytic streptococci in the contents of periodontal pockets are present in quantities not available for detection by the applied method (<2.7 lg CCU/mL). In terms of species, Streptococcus oralis and Streptococcus salivarius are more characteris-tic in gingival crevicular fluid in healthy individuals (93.8% of selected strains). In 68.4 +/- 3.32% of patients in group A, 64.0 +/- 3.43% of patients in group B, and 67.5 +/- 3.76% of patients in group C, the dominant species were Streptococcus gordonii and Streptococcus sanguinis (p<0.01), which increased pathogenic potential as they produce streptolysin-O, inhibit complement activation, bind to fibronectine, actively form biofilms on the surface of tooth enamel and gum epithelial surface, and can act as an initiator of adhesion of periodontal pathogens. The other representatives of the resident microflora of the oral cavity - Stomatococcus mucilaginosus and Veillonella parvula for the patients of group C are also found in periodontal pockets with a significantly lower index of persistence and minimal population level. In the post-covid period, both the population level and the frequency of colonization of periodontal pockets by Staphylo-cocci and beta-hemolytic Streptococci decreases rapidly. For these patient groups, unlike for those that did not suffer from COVID-19, we did not find any case of colonization with Staphylococcus aureus, as well as beta-hemolytic Streptococci and Epidermal staphylococcus were also absent. The most characteristic in the post-covid period is a decrease in the proportion of alpha-hemolytic Streptococci, an increase in the proportion of yeast-like fungi of Candida species, as well as the appearance of a significant number of gram-negative rod-shaped bacteria (Enterobacteria and Pseudomonads). In periodontal patien s, the microbial count is approximately 2 orders of magnitude lower than in those with GP who did not suffer from COVID-19 (p<0.05). Conclusions. The overpassed coronavirus disease due to intensive antibiotic therapy leads to a marked decrease in the number of viable saprophytic microorganisms in the periodontal pockets of patients with GP. In the post-covid period for the patients with GP, there is a decrease in the level of colonization of periodontal pockets by species of resident oral microflora - alpha-hemolytic Streptococci, reduction of resident micro-organism's species, and almost complete disappearance of transient microflora. On the other hand, the frequency of colonization of periodontal pockets by fungi species, enterobacteria, and pseudomonads significantly increases. There are more expressed disorders in the periodontal pocket's microbiome for the patients with a severe and complicated course of coronavirus disease, such as post-covid pulmonary fibrosis, which requires reconsideration of approaches to therapeutic and pharmacological treatment in this category of patients.Copyright © 2022, Zabolotny Institute of Microbiology and Virology, NAS of Ukraine. All rights reserved.

The conundrum of COVID-19 mRNA vaccine-induced anaphylaxis.

Novel messenger RNA (mRNA) vaccines have proven to be effective tools against coronavirus disease 2019, and they have changed the course of the pandemic. However, early reports of mRNA vaccine-induced anaphylaxis resulted in public alarm, contributing toward vaccine hesitancy. Although initial reports were concerning for an unusually high rate of anaphylaxis to the mRNA vaccines, the true incidence is likely comparable with other vaccines. These reactions occurred predominantly in young to middle-aged females, and many had a history of allergies. Although initially thought to be triggered by polyethylene glycol (PEG), lack of reproducibility of these reactions with subsequent dosing and absent PEG sensitization point away from an IgE-mediated PEG allergy in most. PEG skin testing has poor posttest probability and should be reserved for evaluating non-vaccine-related PEG allergy without influencing decisions for subsequent mRNA vaccination. Immunization stress-related response can closely mimic vaccine-induced anaphylaxis and warrants consideration as a potential etiology. Current evidence suggests that many individuals who developed anaphylaxis to the first dose of an mRNA vaccine can likely receive a subsequent dose after careful evaluation. The need to understand these reactions mechanistically remains critical because the mRNA platform is rapidly finding its way into other vaccinations and therapeutics.

Complete response to eculizumab in atypical hemolytic uremic syndrome with homozygous C3 mutation

Case Report: Atypical Hemolytic Uremic Syndrome (atypical HUS) is a rare and severe form of thrombotic microangiopathy (TMA) characterized by thrombocytopenia, intravascular hemolysis, and acute kidney injury with an incidence of 1 per million.1 Dysregulation and overactivation of the complement alternative pathway due to genetic mutations have been detected in 40-60% of patients with sporadic or familial atypical HUS.2,4 Triggers include viral illness, pregnancy, malignancy, sepsis, or sporadically with no known inciting event.1 Atypical HUS is a severe disease with a 2-10% risk of mortality, 33% risk of end-stage renal failure, and 50% chance of relapse.5 A 24-year-old female with prior history of atypical HUS at the age of 16 (with response to plasmapheresis) presented to the ER with a 5-day history of fever, chills, sore throat, nausea, vomiting, and dark urine. She tested positive for COVID-19. The exam revealed scleral icterus and scattered petechiae. Labs demonstrated nadir hemoglobin (Hgb) of 9.2 g/dL, platelet count of 52 000k/uL, haptoglobin < 30 mg/dL, peak LDH 1128U/L and creatinine 4.62 mg/dL. Urinalysis is consistent with hemoglobinuria. Schistocytes were noted on the peripheral smear. Rapid streptococcal antigen test and C3, C4, and IgA levels were unremarkable. Chest X-Ray, X-ray KUB, and ultrasound abdomen were unremarkable. The pregnancy test was negative. ADAMTS13 was >100%. Genetic analysis after the initial episode at age 16 revealed autosomal recessive inheritance c.193A > c gene mutations in C3. The patient received IV fluids, ceftriaxone for cystitis, and two units of Fresh Frozen Plasma. She initiated treatment with eculizumab. She also received the MENVEO and meningitis B vaccine per protocol due to the risk of meningitis from terminal complement deficiencies. After 4 infusions of eculizumab, patient's labs improved to platelet count of 307 000 k/uL, Hgb 12.2 g/ dL (nadir 9.2 g/dL), haptoglobin 78 mg/dL normalization of LDH and improved creatinine. Atypical HUS is a rare form of TMAwith mutations in C3 noted in 5% of cases. Complement cascade dysfunction leads to endothelial deposits and microvasculature damage. The resulting prothrombotic state causes obstructive microvascular thrombi predominantly affecting the kidneys but can cause multiorgan dysfunction. The SARS-CoV-2 virus may precipitate atypical HUS relapse due to endothelial damage and complement activation further intensified in patients with existing complement aberrations. Plasma exchange remains a standard of care for atypical HUS, as it effectively removes the antibodies and other proteins. Eculizumab a humanized monoclonal IgG antibody binds to complement proteins, preventing cleavage into C5a and C5b blocking C5b-9(MAC) activation. In patients with CFH, CFI, C3, and CFB mutations, eculizumab is the preferred intervention. Copyright © 2023 Southern Society for Clinical Investigation.

A Series of Catastrophic Events

Case report - Introduction: Catastrophic antiphospholipid syndrome (CAPS) is a rare, life-threatening disease occurring in up to 1% of antiphospholipid syndrome (APS) cases. It was first defined in 1992 and remains a difficult to treat entity with a mortality rate of 37%. We describe a patient with systemic lupus erythematosus (SLE) and CAPS presenting with simultaneous multi-organ injuries who was successfully managed with 'triple' therapy including cyclophosphamide. Case report - Case description: A 42-year-old female presented to her local hospital with chest pain and worsening vision. She had a background of SLE, triple antibody-positive APS (previous DVT, pregnancy loss and strokes), hypertension, a metallic mitral valve, a previous myocardial infarction and pre-existing visual impairment due to a prior intra-cerebral bleed related to anticoagulation. Examination revealed a faint malar rash, cortical blindness and long tract neurological signs. Her ECG showed ischaemic changes and the admission troponin was significantly raised (3773ng/L). An echocardiogram showed new left ventricular dysfunction and a subsequent cardiac MRI was in keeping with coronary artery disease. Investigations showed an acute kidney injury, newly deranged liver function tests and a raised INR (>11, with no bleeding). Complement was normal with a low dsDNA titre. Urinalysis revealed proteinuria and a protein creatinine ratio measured 176mg/mmol. MRI diffusion weighted brain imaging showed acute bilateral occipital and left fronto-parietal infarcts. She had symptoms of a lupus flare with arthralgia and a butterfly facial rash. COVID-19 PCR tests were negative and she had not been recently vaccinated. She was diagnosed with CAPS and transferred to St Thomas' hospital intensive care. On arrival, she received 1mg intravenous vitamin K followed by triple therapy for CAPS: an unfractionated heparin infusion, oral prednisolone 40mg daily, 5 days of plasma exchange and, given her background of SLE, she was treated with intravenous cyclophosphamide (according to the EUROLUPUS regimen). Intravenous methylprednisolone was avoided due to a previous hypertensive encephalopathy reaction. She responded rapidly. Her troponin fell from a peak of 5054 to 294ng/ L, her creatinine settled at a new baseline (232umol/L) and her liver function normalised. She was switched back to warfarin due to her metallic valve and started on aspirin for cardiovascular secondary prevention. She required physical and occupational therapy due to her strokes but recovered well. Case report - Discussion: According to the 2003 criteria, CAPS can be classified as definite when there is evidence of: -3 organs involved, development of manifestations simultaneously or within a week, confirmation by imaging and/or histopathology of small vessel occlusion and positive antiphospholipid antibodies. Probable CAPS is when 3 out of the 4 criteria are present. In this case, three organs were confirmed to be involved with imaging showing cerebral and cardiac ischaemia. Her creatinine rose from a base of 190 to 289umol/L coupled with a high protein creatinine ratio confirming renal involvement. A Budd-Chiari syndrome was also suspected due to deranged liver function tests and INR, though imaging performed after therapy did not confirm this. A biopsy of any of these four organs was not feasible given the severity of her presentation and coagulopathy. There are no randomised controlled trials but data from the CAPS registry guides treatment and management follows a logical approach: anticoagulation to treat thrombosis, glucocorticoids for inflammation and plasma exchange (or IVIG) to remove the circulating autoantibodies. Triple therapy was associated with a reduced mortality compared to no treatment (28.6% versus 75%, respectively). Following analyses from the CAPS registry we also chose to treat with cyclophosphamide, which is associated with improved survival in patients with SLE. This decision was based on the clinical features of an SLE flare as opposed to serological grounds. There have b en reports of rituximab and eculizumab being used successfully in CAPS, though generally as a last resort. As complement activation is seen in animal models of antiphospholipid syndrome thrombosis and rituximab is often used in refractory SLE, they may prove to be promising agents for refractory CAPS. Case report - Key learning points: 1. Prompt recognition and early treatment is vital in managing CAPS 2. Triple therapy with anticoagulation, glucocorticoids and plasma exchange / IVIG is associated with better survival in CAPS 3. Cyclophosphamide is associated with better survival in patients with CAPS and concomitant SLE.

Pitfalls in complement analysis: A systematic literature review of assessing complement activation.

Background: The complement system is an essential component of our innate defense and plays a vital role in the pathogenesis of many diseases. Assessment of complement activation is critical in monitoring both disease progression and response to therapy. Complement analysis requires accurate and standardized sampling and assay procedures, which has proven to be challenging. Objective: We performed a systematic analysis of the current methods used to assess complement components and reviewed whether the identified studies performed their complement measurements according to the recommended practice regarding pre-analytical sample handling and assay technique. Results are supplemented with own data regarding the assessment of key complement biomarkers to illustrate the importance of accurate sampling and measuring of complement components. Methods: A literature search using the Pubmed/MEDLINE database was performed focusing on studies measuring the key complement components C3, C5 and/or their split products and/or the soluble variant of the terminal C5b-9 complement complex (sTCC) in human blood samples that were published between February 2017 and February 2022. The identified studies were reviewed whether they had used the correct sample type and techniques for their analyses. Results: A total of 92 out of 376 studies were selected for full-text analysis. Forty-five studies (49%) were identified as using the correct sample type and techniques for their complement analyses, while 25 studies (27%) did not use the correct sample type or technique. For 22 studies (24%), it was not specified which sample type was used. Conclusion: A substantial part of the reviewed studies did not use the appropriate sample type for assessing complement activation or did not mention which sample type was used. This deviation from the standardized procedure can lead to misinterpretation of complement biomarker levels and hampers proper comparison of complement measurements between studies. Therefore, this study underlines the necessity of general guidelines for accurate and standardized complement analysis.

Role of the Complement System and Vascular Endothelium in COVID-19 Pathogenesis

Background: Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 infection has become a global pandemic, presenting with varying degrees of severity from respiratory distress to multi-organ damage. Kidneys are of several organs affected in COVID-19, with acute kidney injury (AKI) being a common consequence, occurring in more than 30% of patients with severe COVID-19. While the underlying mechanisms of COVID-19 pathogenesis remain poorly understood, there is evidence linking complement system overactivation and endothelial injury to organ damage that increases the risk of mortality in COVID-19. Evidence from previous coronavirus epidemics also suggest direct involvement of inflammation, complement dysregulation, and endothelial cell dysfunction. Thus, we hypothesize that vascular endothelial injury resulting from complement overactivation contributes to COVID-19-associated organ injury. Method(s): Clinical information and sera from SARS-CoV-2+ patients with mild (n=7) and severe COVID-19 (n=7) diseases were obtained from the Canadian COVID-19 Prospective Cohort Study (CANCOV). Complement activation on ECs was evaluated via immunofluorescence assays, measuring the deposition of complement products C3b and C5b-9 on Human Umbilical Vein Endothelial Cells exposed to control or patient sera. In addition, a permeability assay using a transwell model was used to measure the integrity of the endothelial monolayer exposed to patient sera. Result(s): Complement was found to be overactivated on ECs treated with SARSCoV-2+ patient sera compared to those treated with normal human serum as evidenced by significantly increased C3b and C5b-9 deposition. While ECs treated with sera from patients with mild COVID-19 seemed to have higher C3b deposition, ECs treated with sera from patients with severe COVID-19 disease were associated with higher C5b-9 deposition. In addition, increased permeability of the monolayer incubated with SARSCoV-2+ patient sera was seen over time regardless of disease severity. However, ECs tretaed with severe COVID-19 patient sera had signficiantly increased vascular leakiness as evidenced by increased permeability of the treated monolayer. Conclusion(s): Thus, we conclude that complement is overactivated in SARS-CoV-2+ patients and use of anti-complement therapies may be an effective strategy in treating COVID-19 associated vascular injury, hyperinflammation, and organ damage.

Pseudolung Cancer Lymphadenopathy, Development of Idiopathic Tubulointerstitial Nephritis, and Dense Deposit Disease Following Pfizer-BioNTech COVID-19 Vaccination

Introduction: Despites reports of glomerulonephritis associated with COVID-19 mRNA vaccines, no study has reported about the dense deposit disease (DDD). Here we present a case of pseudolung cancer lymphadenopathy following COVID-19 mRNA vaccine, following which the patient developed idiopathic tubulointerstitial nephritis (TIN) and DDD. Case Description: A 74-year-old man received his second dose of the mRNA vaccine, and he developed fever, urticaria, and dyspnea. On further examination, he had pleural effusion and right hilar lymphadenopathies, which were improved with conservative therapy. On 48 days after the second vaccination, he developed renal dysfunction and new-onset hematuria. Light microscopy findings by a renal biopsy demonstrated apparent mesangial cell proliferation and diffuse inflammatory cell infiltration in the interstitium. Immunofluorescence analysis revealed 1+ positive results for IgG and IgM, negative results for IgA, and 2+ positive results for C3 with a garland pattern on the capillary walls. Electron microscopy detected that continuous and thickened highly dark-stained spotty dense deposits in the glomerular basement membrane. Based on the decrease in C3 and pathological findings, idiopathic TIN accompanied with DDD was diagnosed. Discussion(s): After vaccination acute allergic reaction, pseudolung cancer lymphadenopathy, hematuria, and hypocomplementemia were observed. Thus, both coincidental onset with DDD and TIN following acute allergic response that occurred about 7 weeks before made us think that each event or disease might be associated with COVID-19 mRNA vaccination as part of immunological reactions. In complement activation related pseudoallergy syndrome, it is recently recognized that several modernday therapeutic molecules may activate complements via the nonIgE mediated mechanism with the C3a and C5a anaphylatoxins binding to mast cells, triggering that the release of a number of several vasoactive mediators that cause the clinical features associated with hypersensitivity reactions. mRNA vaccine might have contributed to the development of lymphadenopathies, TIN and DDD in this case. Moreover, TIN and DDD might be associated with the activated alternative pathway induced by the mRNA vaccine.

COVID-19, ACE2, Plus Human Angiotensin II: The Perfect Storm

Background: SARs-CoV-2 causes a disease unlike any we have seen before. The virus is similar to some, but the resultant disease is vastly different in the short and long term. This appears to be due to the Cytokine Storm (CS). The connection between COVID and the CS is likely related to Angiotensin II. Specifically, the Angiotensin II Type 1 Receptor (AT1R) which is regulated via the ACE2 Receptor (ACE2R). The AT1R pathway is the primary pro-inflammatory pathway that promotes endothelial activation, cellular recruitment and differentiation, T and B cell co-stimulation, and complement activation. This would explain the exact findings in the Cytokine Storm. Method(s): Extensive literature search including international publications and presentations as well as clinical experience. Direct clinical observation of the impact of RAAS on pathophysiology in COVID patients. Result(s): The connection between COVID and the ACE2 Receptor (ACE2R) is the best explanation for the CS. COVID induced ACE2R endocytosis, decreased expression, and decreased transcription facilitates unchecked activation of AT1R. In addition to controlling AT1R mediated endothelial inflammation, the ACE2R is also the regulatory mechanism for the Bradykinin mediated coagulation pathway. This would enhance the explanation of not just the inflammation, but the clotting as well. Conclusion(s): The ACE2R and AT1R connection is the best explanation for the tissue damage from COVID. AT1R mediated inflammation and vascular complications would explain the laboratory findings, histology findings, and clinical symptoms found in the Cytokine Storm. Furthermore, one of the key differences between SARS-CoV-2 and other recent virus outbreaks is the affinity with which SARS-CoV-2 binds to the ACE2R. It has the highest binding affinity of all current coronaviruses. This may explain the different outcomes in certain patients also. Any patients that would be at a higher RAAS baseline (Diabetes, Kidney Disease, Heart Disease) would be more likely to have complications. This is what we have seen clinically. Finally, this pathway gives us a mechanism for certain therapies that have shown promise such as Tocilizumab and Baricitinib. It also offers further treatment potentials for RAAS. In my experience, adequate blood pressures and ongoing diuresis are beneficial. Of particular benefit is diuresis with Hypertonic Saline, which has the best RAAS suppression and diuresis capabilities of any diuretic adjunct.

Distinction of early complement classical and lectin pathway activation via quantification of C1s/C1-INH and MASP-1/C1-INH complexes using novel ELISAs.

The most commonly used markers to assess complement activation are split products that are produced through activation of all three pathways and are located downstream of C3. In contrast, C4d derives from the cleavage of C4 and indicates either classical (CP) or lectin pathway (LP) activation. Although C4d is perfectly able to distinguish between CP/LP and alternative pathway (AP) activation, no well-established markers are available to differentiate between early CP and LP activation. Active enzymes of both pathways (C1s/C1r for the CP, MASP-1/MASP-2 for the LP) are regulated by C1 esterase inhibitor (C1-INH) through the formation of covalent complexes. Aim of this study was to develop validated immunoassays detecting C1s/C1-INH and MASP-1/C1-INH complex levels. Measurement of the complexes reveals information about the involvement of the respective pathways in complement-mediated diseases. Two sandwich ELISAs detecting C1s/C1-INH and MASP-1/C1-INH complex were developed and tested thoroughly, and it was investigated whether C1s/C1-INH and MASP-1/C1-INH complexes could serve as markers for either early CP or LP activation. In addition, a reference range for these complexes in healthy adults was defined, and the assays were clinically validated utilizing samples of 414 COVID-19 patients and 96 healthy controls. The immunoassays can reliably measure C1s/C1-INH and MASP-1/C1-INH complex concentrations in EDTA plasma from healthy and diseased individuals. Both complex levels are increased in serum when activated with zymosan, making them suitable markers for early classical and early lectin pathway activation. Furthermore, measurements of C1-INH complexes in 96 healthy adults showed normally distributed C1s/C1-INH complex levels with a physiological concentration of 1846 ± 1060 ng/mL (mean ± 2SD) and right-skewed distribution of MASP-1/C1-INH complex levels with a median concentration of 36.9 (13.18 - 87.89) ng/mL (2.5-97.5 percentile range), while levels of both complexes were increased in COVID-19 patients (p<0.0001). The newly developed assays measure C1-INH complex levels in an accurate way. C1s/C1-INH and MASP-1/C1-INH complexes are suitable markers to assess early classical and lectin pathway activation. An initial reference range was set and first studies showed that these markers have added value for investigating and unraveling complement activation in human disease.

Exposure of the Basophilic Cell Line KU812 to Liposomes Reveals Activation Profiles Associated with Potential Anaphylactic Responses Linked to Physico-Chemical Characteristics.

Lipidic nanoparticles (LNP), particularly liposomes, have been proven to be a successful and versatile platform for intracellular drug delivery for decades. Whilst primarily developed for small molecule delivery, liposomes have recently undergone a renaissance due to their success in vaccination strategies, delivering nucleic acids, in the COVID-19 pandemic. As such, liposomes are increasingly being investigated for the delivery of nucleic acids, beyond mRNA, as non-viral gene delivery vectors. Although not generally considered toxic, liposomes are increasingly shown to not be immunologically inert, which may have advantages in vaccine applications but may limit their use in other conditions where immunological responses may lead to adverse events, particularly those associated with complement activation. We sought to assess a small panel of liposomes varying in a number of physico-chemical characteristics associated with complement activation and inflammatory responses, and examine how basophil-like cells may respond to them. Basophils, as well as other cell types, are involved in the anaphylactic responses to liposomes but are difficult to isolate in sufficient numbers to conduct large scale analysis. Here, we report the use of the human KU812 cell line as a surrogate for primary basophils. Multiple phenotypic markers of activation were assessed, as well as the release of histamine and inflammasome activity within the cells. We found that larger liposomes were more likely to result in KU812 activation, and that non-PEGylated liposomes were potent stimulators of inflammasome activity (four-fold greater IL-1ß secretion than untreated controls), and a lower ratio of cholesterol to lipid was also associated with greater IL-1ß secretion ([Cholesterol:DSPC ratio] 1:10; 0.35 pg/mL IL-1ß vs. 5:10; 0.1 pg/mL). Additionally, PEGylation appeared to be associated with direct KU812 activation. These results suggest possible mechanisms related to the consequences of complement activation that may be underpinned by basophilic cells, in addition to other immune cell types. Investigation of the mechanisms behind these responses, and their impact on use in vivo, are now warranted.

B Cells Induce Hypertension, AT1AA, And Complement Activation In Preeclampsia Or In Response To A Prior Covid-19 Infection During Pregnancy

Preeclampsia (PE), new onset hypertension (HTN) during pregnancy, is associated with placental ischemia and chronic inflammation that includes increased CD4+ T cells, B cells secreting agonistic autoantibodies against the angiotensin II type 1 receptor (AT1AA), and activation of the complement system. Previous studies have shown AT1-AA is produced in patients with COVID-19 infection. Interestingly, having had COVID-19 during pregnancy is associated with increased incidence of developing a PE phenotype during pregnancy. We have previously shown an important role for B cell depletion or AT1AA inhibition to attenuate HTN in rat models of PE. Collectively, this data suggests B cells contribute to PE development and that B cells may increase incidence of PE in patients with a history (Hx) of COVID-19 during pregnancy through production of the AT1AA. We hypothesize B cells from PE or CV Hx PE patients produce AT1AA resulting in HTN and complement activation in pregnancy. Placental B cells were isolated from normal pregnant (NP), PE, normotensive (NT) CV Hx, or PE CV Hx patients at delivery. B cells were transferred i.p. into pregnant athymic rats at gestation (GD) 12. On GD18, carotid catheters were inserted. On GD19, blood pressure was measured and tissues collected. PE B cell recipients had increased Mean Arterial Pressure (MAP) (115+/-3 mmHg n=6) compared to NP B cell recipients (97+/-4 mmHg n=6 p<0.05). PE B cell recipients had increased AT1AA (20+/-2 DELTABPM n=4) compared to NP B cell recipients (6+/-1 DELTABPM n=4 p<0.05). PE B cell recipients had increased markers of complement activation such as reduced plasma C4 (1302+/-169 mug/mL n=4) and C3 (516+/-45 mug/mL n=4) compared to recipients of NP B cells (2348+/-338 mug/mL n=4 p<0.05) and (790+/-66 mug/mL n=4 p<0.05) respectively. CV Hx PE B cell recipients had elevated MAP (108+/-3 mmHg n=4) compared to CV Hx NT B cell recipients (101+/-7 mmHg n=4) and increased AT1AA (24+/-3 DELTABPM n=3) compared to CV Hx NT B cell Recipients (4+/-1 DELTABPM n=4 p<0.05). Collectively, this study demonstrates an important role for B cells to cause HTN during pregnancy;and indicates that B cells contribute to a higher incidence of PE in women with a Hx of CV infection during pregnancy possibly by secreting AT1-AA.

MACROGLOSSIA IN THE SETTING OF COVID-19

SESSION TITLE: COVID-19 Case Report Posters 1 SESSION TYPE: Case Report Posters PRESENTED ON: 10/17/2022 12:15 pm - 01:15 pm INTRODUCTION: Macroglossia is a rare but life-threatening symptom that disrupts a person's ability to talk, swallow, and can also compromise their airway. Although not very well studied, there are several case reports describing a possible association between COVID-19 infection and macroglossia in people with African ancestry. We present an African American man who developed significant macroglossia several days after testing positive for COVID-19. CASE PRESENTATION: A 59 y/o African American male with a history of chronic bronchitis and tobacco use presented with 4 days of dyspnea. Sars-Cov-2 PCR was positive. Chest x-ray revealed bilateral, diffuse lung infiltrates. He had an elevated CRP of 295 and a d-dimer of 265. He became lethargic and hypercapnic requiring intubation which was nontraumatic. He was sedated, paralyzed, and proned. He received steroid therapy, broad spectrum antibiotics and a dose of Sarilumab. About a week later, he developed macroglossia that worsened over the course of days. Side effect profiles of each of his medications did not reveal any increased likelihood of macroglossia. C1Q complement cascade was mildly elevated and C1 esterase inhibitor level was normal. Diagnosis and treatment was necessary at this point as concerns for tongue central necrosis were raised and baseline tongue size would be required for proper evaluation and surgical intervention if necessary. He was given 4 units of FFP for possible angioedema with no improvement. CT Neck W/ contrast revealed edema and protrusion of the tongue without a discrete mass. Workup for acromegaly, sarcoidosis, amyloidosis, and hypothyroidism were negative. A pressure ulcer developed on his tongue due to the endotracheal tube and so he underwent tracheostomy. His tongue was draped in Chlorhexidine soaked gauze as well as Vashe wound solution. As he recovered from COVID-19 pneumonia, his respiratory status improved as well as his macroglossia. His tracheostomy was decannulated and his tongue returned to its baseline size. DISCUSSION: Macroglossia can lead to complications including airway compromise, dysphagia, or speech difficulties. It has been heavily proposed in the literature that COVID-19 infection can lead to postinfectious inflammatory peripheral nerve injury secondary to immune driven mechanisms. It was also previously proposed in literature based on immune-histochemical analysis of a tongue tissue sample taken from a COVID-19 patient that tongue muscle atrophy occurs as well as macrophage infiltration similar to that of nerve injury repair which can eventually lead to macroglossia. CONCLUSIONS: As the effects of COVID-19 are becoming better studied overtime, macroglossia, especially in those with African ancestry, is increasingly coming under the radar. This case report seeks to educate clinicians on this possible sequela and encourage supportive treatment in hopes that the tongue will recover. Reference #1: McCrossan S, Martin S, Hill C. Tongue Reduction for Macroglossia. J Craniofac Surg. 2021;32(5):1856-1859. doi:10.1097/SCS.0000000000007276 Reference #2: Colombo D, Del Nonno F, Nardacci R, Falasca L. May macroglossia in COVID-19 be related not only to angioedema?. J Infect Public Health. 2022;15(1):112-115. doi:10.1016/j.jiph.2021.10.026 Reference #3: Fernandez CE, Franz CK, Ko JH, et al. Imaging Review of Peripheral Nerve Injuries in Patients with COVID-19. Radiology. 2020;298 (3). https://doi.org/10.1148/radiol.2020203116 DISCLOSURES: No relevant relationships by Megan Devine No relevant relationships by Devin Haney No relevant relationships by Es-Haq Hassanin No relevant relationships by Nadim Islam No relevant relationships by Alyssa Weyer