Dermatomyositis with Specific Autoantibodies Appearing after Sars-Cov-2 Infection or Mrna Vaccination: Hints at a Shared Pathogenetic Mechanism

Background. Environmental factors such as infections and vaccines are known to trigger dermatomyositis (DM), and during the recent SARS-CoV-2 pandemic this has become even clearer. SARS-CoV-2 infection may share features with anti-MDA5 DM, such as rapidly progressive lung involvement, cutaneous lesions and cytokine release syndrome. A few case reports of DM following SARSCoV-2 vaccination have been published, suggesting the onset of an aberrant immune response leading to DM with specific autoantibody signatures and severe organ impairment. Methods. Clinical and laboratory data of the 2 case reports were obtained from electronic clinical charts in Humanitas Research Hospital (Rozzano, Milan, Italy). Autoantibody analysis was performed by protein-immunoprecipitation for anti-MDA5 and immunoblot for anti-Ro52 and TIF1gamma antibodies as per protocol. Results. Case report 1 is a 71-year-old woman who developed fever, cough, and anosmia, which resolved spontaneously in two weeks, but did not undergo a nasopharyngeal swab, while her relatives were diagnosed with SARS-CoV-2 infection. When symptoms improved, she developed arthralgia and skin lesions on her face, chest, and hands for which she started topical treatment, with negative SARSCoV-2 nasopharyngeal swab and positive serum test for IgG against SARS-CoV-2 spike protein. For the persistence of the skin rash and arthralgia, she was admitted to our Department in March 2021. Blood tests showed mild elevation of C reactive protein (2.1 mg/L -normal value NV<5), aspartate (84 UI/L) and alanine aminotransferase (133 UI/L -NV<35), ferritin (595 ng/ml -NV<306), troponin I (19 ng/L -NV<14), and BNP (251 pg/ml -NV<100) with normal complete blood cell count, creatine kinase, C3 and C4. IgG antibodies for SARS-CoV-2 spike protein were confirmed to be elevated (96 AU/ml -NV<15). Autoantibodies associated with connective tissue diseases were tested and only anti-MDA5 antibodies were positive at immunoprecipitation. A punch biopsy of a Gottron-like lesion on the left hand showed leukocytoclastic vasculitis. We observed reduced capillary density with neoangiogenesis and ectasic capillaries at the nailfold capillaroscopy. EKG and ecocardiography were normal, while cardiac magnetic resonance detected abnormalities in the parametric sequences, consistent with signs of previous myocarditis. A lung CT scan revealed pulmonary emphysema while respiratory function tests demonstrated reduced volumes (FVC 82%, FEV1 64%, inadequate compliance CO diffusion test). Based on the biochemical and clinical findings, a diagnosis of anti-MDA5-associated DM with skin and heart involvement was made and treatment with low-dose methylprednisolone (0.25 mg/kg daily) and azathioprine 100 mg was started, then switched to mycophenolate because not effective on skin lesions. Case report 2 is an 84-year-old woman with history of colon cancer (surgical treatment) and oral lichen treated with low doses steroids in the last 2 years. After the 2nd dose of SARS-CoV-2 mRNA vaccination, in March 2021 she developed skin rash with V-sign, Gottron's papules, periungueal ulcers, muscle weakness and fatigue, thus she performed a rheumatologic evaluation. Blood tests showed mild elevation of creatine kinase (484 UI/L, NV <167), CK-MB (9.6ng/ml, NV <3.4), BNP (215 pg/ml -NV<100) with normal values of complete blood cell count, C3 and C4. Anti-Ro52kDa and TIF1gamma were positive at immunoblot, thus we confirmed a diagnosis of DM. The clinical evaluation also showed active scleroderma pattern at nailfold capillaroscopy, normal echocardiography, bronchiectasia but not interstitial lung disease at lung CT, and normal respiratory function tests (FVC 99%, FEV1 99%, DLCO 63%, DLCO/VA 81%). A PET-CT scan was performed to exclude paraneoplastic DM, and treatment with steroids and mycophenolate was started. Conclusions. SARS-CoV-2 may induce mechanisms for escaping the innate immunity surveillance and causing autoimmune diseases, but more clinical and functional studies are needed to demonstrate this possible association.

Unusual Myositis of the Temporalis Muscle Occurring with Acute Covid-19

Background. SARS-CoV-2 infection can be accompanied by neuromuscular disorders. Rhabdomyolysis and Guillain-Barre syndrome have been described repeatedly. There are case reports of inflammatory myopathies manifesting during COVID-19, presenting as dermatomyositis, polymyositis or immune-mediated necrotizing myopathy, with dermatomyositis-like presentations most commonly reported. Larger cases series are from postmortem examinations of COVID-19 patients, where variable inflammatory pathology of the skeletal muscle has been found frequently but without local detection of the actual virus. Thus, autoimmune mechanisms or the systemic interferon response are discussed as causes. We report a case of focal inflammatory myopathy with perimysial pathology of the temporalis muscle occurring with acute, but mild COVID-19. Methods. Case report of clinical observations, cranial MRI, histopathological, and laboratory findings. 3T cranial MRI was performed with gadolinium contrast. Open temporalis muscle biopsy was performed. The sample underwent standard cryohistological studies as well as immunohistochemistry with antibodies against MHC-I and II, CD3, CD4, CD19, CD68, anti-MAC, p62 and MxA. Testing for auto-antibodies was based on immunoblots or ELISA. RT-PCR for SARS-CoV-2 was run with RNA extracted from cryopreserved muscle. Results. A Caucasian woman in her 60s with no history of autoimmune or muscle complaints developed swelling and pain of the right jaw musculature five days after testing positive for SARS-CoV-2 due to respiratory tract symptoms. In addition, she experienced trismus, but no further neuromuscular complaints. The course of respiratory tract symptoms stayed mild. She had been vaccinated previously with single shot SARS-CoV-2 vector vaccine. Due to persistent swelling and complaints, giant cells arteritis was excluded by unresponsiveness to five days oral steroids and sonography of the temporal artery. Cranial MRI was performed nearly four weeks after the SARS-CoV-2 infection and showed marked swelling and oedema of the temporalis muscle. Its biopsy showed numerous CD68 and acid phosphatase positive cells infiltrating from perimysial perivascular foci towards the endomysium with perimysial damage but little damage of adjacent, perifascicular muscle fibres. Muscle fibres did not react with anti-MHC-II, anti-MAC or -MxA. Capillaries did not react with anti-MAC or -MxA. SARS-CoV-2 RNA was not detected in muscle tissue. Serum creatine kinase was not elevated in the subacute phase. Slightly elevated ANA titre led to detection of autoantibodies against proliferating cell nuclear antigen (PCNA). No pathological results for other autoantibodies, including myositis-specific antibodies and anti-ds-DNA, were found in blood. Neither were antibodies against hepatitis C and B viruses. Retesting 15 weeks after infection, anti-PCNA immunoblot was still positive, but ELISA did not indicate a pathologic titre. The swelling, myalgia and trismus regressed spontaneously a month after onset, yet the latter still persists at the time of reporting. Conclusion. Our case diverges from the majority of COVID-19 associated my-ositis reports in the unusual location of the focal myositis and the histopathological pattern of predominantly perimysial damage and histiocytic infiltration. It concurs with the literature as no SARS-CoV2 RNA could be detected in the muscle. Anti-PCNA is associated very rarely with myositis. Other associated disorder (systemic lupus erythematosus, chronic viral hepatitis B or C) were not found. Increased levels of autoantibodies are reported in COVID-19 and mostly attributed to loss of self-tolerance during the acute disease phase. Interestingly, the structural protein M of SARS-CoV-2 appears to interact notably with PCNA in infected cells. Still, the causal connection between the myositis and COVID-19 in this case is based on the close temporal association in the absence of alternative, competing explanations from the medical history and findings.

ENDOTHELIAL INFLAMMATION AND DYSFUNCTION INDUCED BY SARS-COV-2 SPIKE PROTEIN 1 INVOLVE ADAM17 AND INTERFERON ACTIVATION PATHWAYS

Objective: COVID19 is associated with vascular inflammation. IFN-alpha (IFNa) and IFN-lambda3 (IFNl3) are potent cytokines produced in viral infections. Their effects involve interferon-stimulated genes (ISGs) and may influence expression of angiotensin-converting enzyme 2 (ACE2), the receptor for S-protein (S1P) of SARS-CoV-2. We hypothesized that S1P-induced immune/inflammatory responses in endothelial cells (EC) are mediated via IFN-activated pathways Design and methods: Human ECs were stimulated with S1P (1 mg/mL), IFNa (100ng/mL) or IFNl3 (100IU/mL). Because ACE2, ADAM17 and TMPRSS2 are important for SARS-CoV-2 infection, we used inhibitors of ADAM17 (marimastat, 3.8 nM), ACE2 (MLN4760, 440pM), and TMPRSS2 (camostat, 50 mM). Gene and protein expression was investigated by real-time PCR and immunoblotting, respectively. Vascular function was assessed in mesenteric arteries from wild-type (WT) normotensive and hypertensive (LinA3) mice and in ISG15-deficient (ISG15KO) mice. Results: S1P increased expression of IFNa (3-fold), IFNl3 (4-fold) and ISGs (2-fold) in EC (p < 0.05). EC responses to IFNa (ISG15: 16-fold) were greater than to IFNl3 (ISG15: 1.7-fold) (p < 0.05). S1P increased gene expression of IL-6 (1.3-fold), TNFa (6.2-fold) and IL-1b (3.3-fold), effects that were amplified by IFNs. Only the ADAM17 inhibitor marimastat inhibited S1P effects. IFNa and IFNl3 increase protein expression of ADAM17 (27%) and TMPRSS2 (38%). No changes were observed on ACE2 expression. This was associated with increased phosphorylation of Stat1 (134%), Stat2 (102%), ERK1/2 (42%). EC production of IL-6 was increased by IFNa (1,230pg/mL) and IFNl3 (1,124pg/mL) vs control (591pg/mL). Nitric oxide generation and eNOS phosphorylation (Ser1177) were reduced by IFNa (40%) and IFNl3 (40%). Vascular functional responses demonstrated that endothelium-dependent vasorelaxation (% Emax) in vessels from WT-mice stimulated with IFNa (67%) and IFNl3 (71%) were reduced vs control (82%) (p < 0.05). Responses were not altered in vessels from ISG15KO mice. Increased contraction was observed only in vessels from hypertensive mice treated with IFNa (9.1 ± 0.5mN vs control: 7.3 ± 0.3mN) (p < 0.05). Conclusions: In ECs, S1P, IFNa and IFNl3 increased ISG15 and IL-6 by mechanisms dependent on ADAM17. IFNs amplifies endothelial cell inflammatory responses and induced vascular dysfunction through ISG15-dependent mechanisms, with augmented effects in hypertension. Our findings demonstrate that S1P induces immune/inflammatory responses that may be important in endotheliitis associated with COVID-19. This may be especially important in the presence of cardiovascular risk factors, including hypertension.

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

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

Immunogenicity evaluation of Gam-COVID-Vac (Sputnik V)

In November 2020, the Armed Forces of the Russian Federation began mass immunisation of the personnel with Gam-COVID-Vac (Sputnik V), the first Russia vaccine against the new coronavirus infection (COVID-19). Thus, it became necessary to assess post-vaccination antibody levels and the duration and intensity of humoral immunity to COVID-19. The aim of the study was to investigate the immunogenicity and efficacy of Gam-COVID-Vac in military medical staff after vaccination. Materials and methods: the authors determined the presence of specific antibodies in the serum of individuals immunised with Gam-COVID-Vac (477 volunteers) and COVID-19 convalescents (73 patients), using virus neutralisation (VN), enzyme-linked immunosorbent assay (ELISA) with reagent kits by several manufacturers, and immunoblotting. The results of the study were evaluated using analysis of variance. Results: VN detected virus neutralising antibodies in 90.7% of vaccinated subjects;ELISA, in 95.4%. Both VN and ELISA showed lower antibody levels in the vaccinated over 50 years of age. ELISA demonstrated a significantly higher concentration of anti-SARS-CoV-2 spike IgG in the Gam-COVID-Vac group than in the COVID-19 convalescent group. The correlation between antibody detection results by VN and ELISA was the strongest when the authors used their experimental reagent kit for quantitative detection of virus neutralising antibodies by competitive ELISA with the recombinant human ACE2 receptor. Having analysed the time course of neutralising antibody titres, the authors noted a significant, more than two-fold decrease in geometric means of the titres three months after administration of the second vaccine component. Conclusions: the subjects vaccinated with Gam-COVID-Vac gain effective humoral immunity to COVID-19. The decrease in titres indicates the need for revaccination in 6 months.

Dysfunctional hemostasis in patients under extracorporeal life support. A rapid diagnostic approach with therapeutical guidance intentions

Background: Short-term cardiopulmonary extracorporeal life supports (ECLS) are invasive devices whose use has increased exponentially during the COVID-19 pandemic. Major bleeding is a main cause of morbi-mortality in ECLS patients and acquired von Willebrand disease (aVWD) could justify this complication. Aim(s): We aim at investigating the primary hemostasis alterations profile in ECLS patients, and to propose a potential treatment if bleeding. Method(s): Patients in ECLS at our center since June 2021 were included (n = 25). Primary hemostasis was evaluated by: Von Willebrand Factor antigen (VWF:Ag) and activity (VWF:GPIbM) measurement (immunoturbidimetry), VWF multimeric analysis (agarose-gels and immunoblotting), platelet function analysis (PFA-200), and platelet activation (CD62P and CD63 expression by flow cytometry). Studies were performed 24 h after implant, each 7 days, and in the first week after ECLS extraction. T-TAS was used for hemostasis analysis in samples from bleeding patients, before and after in vitro addition of purified VWF. This study was approved by the Hospital Clinic's Ethics Committee (HCB/2021/0200). Result(s): After 24 h of ECLS implant, increased VWF:Ag levels and prolonged PFA occlusion times. In 60% of patients, altered VWF:GPIbM/VWF:Ag ratio ( < 0.7) and loss of VWF high molecular weight multimers (HMWM) were observed. CD62P expression was slightly higher in ECLS patients platelets than in controls (MFI+/-SD of 4.34 +/- 2.2 vs. 3.27 +/- 0.6, respectively;p = 0.3). Early after ECLS extraction, there was normalization of the VWF multimeric profile and PFA values. Interestingly, in samples from bleeding patients, addition of purified VWF reduced significantly the T-TAS occlusion times (776 s+/-207 s vs. 1161 s+/-251 s, Mean+/-SD, post vs. pre, respectively;p = 0.033). Conclusion(s): ECLS caused primary hemostasis alterations, leading to aVWD and platelet activation, solved early after support removal. Hemostatic efficiency in ECLS bleeding patients, with lack of HMWM, was corrected in vitro by providing functional purified VWF. (Figure Presented).

Angiostatin and plasminogen levels as risk factors for mortality in hospitalized Covid-19 patients

Background: Covid-19- associated thrombotic complications are in part due to loss of plasminogen-associated fibrinolytic activity. Angiostatin is a break-down product of plasmin(ogen) with pro-apoptotic properties under hypoxia and acidosis, conditions associated with Covid-19. Hence, it may contribute to fatal outcome in Covid-19. Aim(s): To assess the relationship between plasminogen/angiostatin levels and mortality in hospitalized Covid-19 patients. Method(s): Age-and sex-matched plasma samples (n = 120) from Covid-19 survivors and non-survivors were obtained from Alberta Precision Laboratories (Canada). Angiostatin and plasminogen were quantified by immunoblot. Cox's proportional hazards regression model was used to determine the association between angiostatin/plasminogen levels and risk of death. Hazard ratios [HR] and 95% confidence intervals [CI] were estimated adjusting for time of sample collection (days from hospitalization to collection) and comorbidities (Charlson comorbidity score). The interaction between plasminogen/angiostatin levels and time of sample collection (before or after 7 days from hospital admission) was assessed and stratified HRs were estimated. Result(s): Overall, compared to survivors, non-survivors had a lower plasminogen level (mean+/-SD: 6.6 +/- 2.1 and 5.6 +/- 2.2 arbitrary units). Increase in plasminogen level was significantly associated with reduction in risk of death, independent of time of sample collection and comorbidities (Table 1). When stratified by time of sample collection, the association remained significant only for samples drawn within 7 days of admission (Table 2). Conversely, an overall difference was not observed in angiostatin concentrations (mean+/-SD: 136.0 +/- 42.5 and 143.0 +/- 48.9 mug/ml), and it was not associated with survival outcome (Table 1). However, when stratified by time of collection and adjusted for comorbidity burden, angiostatin was significantly associated with an increased risk of death for samples collected after 7 days from admission (Table 2). Conclusion(s): Both angiostatin and plasminogen were associated with fatal outcome in Covid-19, independent of comorbidities. These associations depended on the time from hospital admission to sample collection and likely reflect dynamic changes in the plasminogen-angiostatin generation system associated with Covid-19 disease progression. (Table Presented).

Unraveling the pathophysiological role of angiostatin in COVID-19

Background: Angiostatin is a break-down product of plasmin(ogen). Physiologically, angiostatin is generated by platelets in an urokinase (uPA)-dependent manner. During normoxia angiostatin has anti-angiogenic/ anti-inflammatory effects and protects against lung injury, however during hypoxia/acidosis it is pro-apoptotic. In SARS-CoV infected mice, the uPA-plasminogen pathway was shown to be the most transcriptionally enriched regulating sublethal vs. lethal infection. Similarly, uPA has been shown to be transcriptionally upregulated in SARS-CoV- 2;however, the role of angiostatin has not been investigated. Aim(s): To assess role of angiostatin in COVID-19. Method(s): Plasma samples from COVID-negative controls and from hospitalized COVID-19 patients (n = 30) were collected (day 1, 7, 14, 28, 70) via the COVID-19 Surveillance Collaboration study. WHO clinical progression scale was used to assess COVID-19 severity. Angiostatin and plasminogen were quantified by immunoblot. VeroE6 cells were infected with SARS-CoV- 2 and treated with angiostatin (140 mug/ml) for 24h at pH 7.5 or 6.9. Cell death was quantified by both TUNEL and the percentage of detached cells. Immunofluorescent staining against the spike protein was used to confirm cellular infection. Result(s): Plasma angiostatin level was elevated in COVID-19 patients compared to COVID-negative controls at baseline. Both angiostatin and plasminogen increased with time of hospitalization in patients with severe COVID-19, but not with mild-to- moderate disease (p = 0.05;Fig.1). In preliminary cell culture experiments, at pH = 7.5 angiostatin decreased the percentage of detached (26 +/- 8% vs 67 +/- 5%;p = 0.0004) and TUNEL-positive VeroE6 (6 +/- 6 vs 11 +/- 7%;p = 0.07) following infection. Conversely, at pH = 6.9 angiostatin increased the percentage of detached cells following infection. Interestingly, angiostatin lowered the percentage of spike protein-positive Vero E6 at both pH (Fig.2). Conclusion(s): Angiostatin concentrations increase with disease progression in severe COVID-19. This likely reflects angiostatin's complex role in COVID-19 pathophysiology. Angiostatin promotes cell death in acidotic microenvironments (associated with severe Covid-19). Conversely, at physiological pH, angiostatin may have protective effects possibly by reducing viral entry and/or replication. (Figure Presented).

THE ROLE of MOLECULAR MIMICRY in SARS-COV-2 RELATED AUTOIMMUNE RHEUMATIC DISEASES

Background: Several studies suggested that SARS-CoV-2 infection may induce autoantibodies related to autoimmune rheumatic diseases (ARD). Objectives: To determine whether polyclonal antibodies from SARS-CoV-2 unin-fected patients with ARDs cross-react with SARS-CoV-2 and vice versa. Methods: 90 sera positive at high-titres for 23 common autoantibodies (all sera stored before 2018), were tested for reactivity against proteins of SARS-CoV-2 (spike protein S1, nucleocapsid NC etc) by ELISA and CMIA. Vice versa, 10 monoclonal antibodies against S1 protein (most of them against RBD) were tested for autoantibody reactivity by indirect immunofuorescence, ELISA, immunoblot and dot/line immunoassays coated with different antigens. Ten post-COVID sera with high titers of anti-Spike abs were tested by ELISAs for reactivity against various autoantigens related to ARDs. Results: 88 out of 90 samples (%), were totally unreactive to SARS-CoV-2 proteins;2 sera, one anti-CCP and one anti-CENP reacted against S protein. All sera tested negative for neutralized abs against SARS-CoV-2. None of 10 sera from SARS-CoV-2 infected patients reacted with different autoantigens by molecular assays. None of the 10 monoclonal abs against S1 protein reacted with 23 different self-antigens. On HEp2 cells as substrate for IIF, 3 of the 10 monoclonal abs gave a low-titre coarse speckled pattern. No reactivity was found by IIFL using tissue substrates. Conclusion: Our data do not suggest a dominant role for molecular mimicry and immunological cross reactivity as a trigger of autoantibodies related to ARDs.

CASE REPORT of A HARD to TREAT DERMATOMYOSITIS after A COVID-19 INFECTION and SUBSEQUENT VACCINATION

Background: A 50 years old woman, a medical doctor, came to our department with symmetrical proximal muscular weakness, several months after Covid-19 infection and three weeks after a second dose of Covid-19 mRNA vaccine. The patient had no prior or family history of autoimmune diseases and take no medicines. In the past she undergone an operation for double-kidney with frequent urinary infections. Objective fndings have shown symmetrical proximal muscular weakness and classic sings of dermatomyositis-Gottron's papules, shawl and holster signs, periungual vasculitis. Objectives: We present a case of a 50 old woman with clinical and laboratory proven dermatomyositis, starting three weeks after a second dose of a Covid1-19 mRNA vaccine without other reasons. Methods: The laboratory tests showed elevated CPK, lactate dehydroge-nase, aspartate aminotransferase and alanine aminotransferase, high ANA-1:1280 and myositis specifc autoantibodies-anti-NXP2 and anti-Mi-2-beta. The electromyography showed myopathic changes and the muscle MRI-symmetrical edema of mm.obturator and mm.adductor brevis. We exclude diseases that may mimic infammatory myopathies. We made a cancer screening-whole body MRI, colonoscopy, gastroscopy, mammography and gynecological exam, immunoblot for detection of paraneoplastic syndrome-associated neuronal antibodies, with no detection of cancer. Muscle biopsy of m.vastus lateralis showed attenuating muscle infammation with advancing muscle atrophy and fbrosis. Results: The diagnose dermatomyositis was made according Bohan and Peter criteria and we start a high dose (1mg/kg/day) glucocorticoid therapy with good initial clinical and laboratory effect. Two months after starting a therapy muscle weakness worsened together with difficulty of swallowing. We excluded steroid myopathy after second EMG and lack of improvement when tapering the GS dose. Methotrexate 20 mg/weekly was added as a steroid sparing drug with good response, but was stopped because fare of pyelonephritis. Accordning to the opinion of dermatologist hydroxychloroquine was started for a couple of weeks, because of active skin manifestations. Muscle weakness worsened on the background of treatment, which was stopped. We started a therapy with intravenous immunoglobulins and considered therapy with cyclophosphamide or azathio-prine after urinary infection. Because the patient was infected for a second time with covid-19, although vaccine, we continued only with glucocorticoids and anti-osteoporotic therapy. Conclusion: The etiology and pathogenesis of infammatory myopathies are not fully clarifed so far. We speculate that the infection with Covid-19 as well as mRNA vaccine trigger infammatory myopathy and compromise the patient's immunity for poor treatment response with glucocorticoids and immunosuppres-sives. On the other hand advanced muscle atrophy and fbrosis within a short period show that suspected triggering factors could be a reason for difficult to treat such type of dermatomyiositis.

Serum levels of IGF system proteins change with the severity of COVID-19

IGF (Insulin-like Growth Factor) system proteins, including their ligands IGF1 and IGF2, their receptor IGF1R and binding proteins (IGFBPs) that control their bioavailability, are involved in pulmonary homeostasis and in respiratory diseases, including COVID-19. However, their circulating levels have not yet been studied comparatively between groups of patients with different degrees of severity of the disease in order to determine their possible value as biomarkers in this context. Serum levels of IGF1, IGF2 and IGF1R were determined by ELISA, and those of IGFBP2, IGFBP3, IGFBP4 and IGFBP5 by immunoblotting. Results were compared between three groups of patients with different degrees of severity of COVID-19, and with those of an uninfected control group (total n = 120): uninfected (n = 24), asymptomatic (n = 32), hospitalized (n = 32) and ICU (n = 32) controls. IGFBP3/IGFBP2 ratios were also quantified. While IGF1 and IGF2 levels decreased in hospitalized and ICU patients, IGF1R levels were increased in ICU patients. IGFBP2 levels were also elevated in ICU patients, and conversely, IGFBP3 and IGFBP5 levels and IGFBP3/IGFBP2 ratios tend to decrease progressively with the severity of the disease. IGFBP4 levels were only significantly increased in the hospitalized patients compared to the control group. Changes in concentration levels of IGF1, IGF2, IGFBP3, and IGFBP5 follow similar patterns with a downward trend with COVID-19 severity, and are opposite to those of IGF1R and IGFBP2. IGFBP4 shows a different profile, being higher in hospitalized patients. Serum levels of IGFs change with the degree of COVID-19 and decreasing IGFBP3 and IGFBP5 levels and IGFBP3/IGFBP2 ratios show up as candidate biomarkers of disease severity.

IMMUNE DYSREGULATION INDUCED BY SPIKE PROTEIN 1 OF SARS-COV-2 INCREASES ENDOTHELIAL CELL DYSFUNCTION VIA TYPE I AND TYPE III INTERFERON ACTIVATION PATHWAYS

Objective: COVID19-associated immunopathology is associated with increased production of interferon (IFN)-alpha (IFNα) and lambda3 (IFNL3). Effects of IFNs are mediated by interferon-stimulated genes (ISGs) and influence expression of angiotensin-converting enzyme 2 (ACE2), the receptor for S-protein (S1P) of SARS-CoV-2. Increasing evidence indicates vascular inflammation in cardiovascular sequelae of COVID19. We hypothesized that S1P-induced immune/inflammatory responses in endothelial cells (EC) are mediated via IFNα and IFNL3. Design and method: Human ECs were stimulated with S1P (1 μg/mL), IFNα (100ng/mL) or IFNL3 (100IU/mL). Because ACE2, metalloproteinase domain-17 (ADAM17) and type-II transmembrane serine protease (TMPRSS2) are important for SARS-CoV-2 infection, cells were treated with inhibitors of ADAM17 (marimastat, 3.8 nM), ACE2 (MLN4760, 440pM), and TMPRSS2 (camostat, 50 μM). Gene and protein expression was investigated by real-time PCR immunoblotting, respectively. Vascular function was assessed in mesenteric arteries from wild-type (WT) normotensive and hypertensive mice and in ISG15-deficient (ISG15KO) mice. Results: EC stimulated with S1P increased expression of IFNα (3-fold), IFNL3 (4-fold) and ISG (2-fold)(p < 0.05). EC exhibited higher responses to IFNα (ISG15: 16-fold) than to IFNL3 (ISG15: 1.7-fold)(p < 0.05). S1P increased gene expression of IL-6 (1.3-fold), TNFα (6.2-fold) and IL-1β (3.3-fold), effects that were maximized by IFNs. Only marimastat inhibited S1P effects. IL-6 was increased by IFNα (1,230pg/mL) and IFNL3 (1,124pg/mL) vs control (591pg/ mL). This was associated with increased phosphorylation of Stat1 (134%), Stat2 (102%), ERK1/2 (42%). Nitric oxide production and eNOS phosphorylation (Ser1177) were reduced by IFNα and (40%) and IFNL3 (40%). Reduced endothelium relaxation maximal response (%Emax) was observed in vessels from WTmice stimulated with IFNα (67%) and IFNL3 (71%) vs control (82%)(p < 0.05) but not in vessels from ISG15KO mice. Increased contraction was observed only in vessels from hypertensive mice treated with IFNα (9.1 ± 0.5mN vs control: 7.3 ± 0.3mN, p < 0.05). Conclusions: In ECs, S1P, IFNα and IFNL3 increased ISG15 and IL-6, processes that involve ADAM17. Inflammation induced by S1P was amplified by IFNs. IFNs induce vascular dysfunction through ISG15-dependent mechanisms, with augmented effects in hypertension. Our findings demonstrate that S1P induces immune/inflammatory responses that may be important in endotheliitis associated with COVID-19. This is especially important in the presence of cardiovascular risk factors, including hypertension.

Hypercapnia Increases ACE2 Protein Expression and Pseudo-SARSCoV- 2 Virus Entry in Airway Epithelial Cells by Augmenting Cellular Cholesterol

Rationale: Individuals with COPD who develop COVID-19 are at increased risk of hospitalization, ICU admission and death. COPD is associated with increased airway epithelial expression of ACE2, the receptor mediating SARS-CoV-2 entry into cells. Hypercapnia commonly develops in advanced COPD and is associated with frequent and potentially fatal pulmonary infections. We previously reported that hypercapnia increases viral replication, lung injury and mortality in mice infected with influenza A virus. Also, global gene expression profiling of primary human bronchial epithelial (HBE) cells showed that elevated CO2 upregulates expression of cholesterol biosynthesis genes, including HMGCS1, and downregulates ATP-binding cassette (ABC) transporters that promote cholesterol efflux. Given that cellular cholesterol is important for entry of viruses into cells, in the current study we assessed the impact of hypercapnia on regulation of cellular cholesterol levels, and resultant effects on expression of ACE2 and entry of Pseudo-SARS-CoV-2 in cultured HBE, BEAS-2B and VERO cells, and airway epithelium of mice. Methods: Differentiated HBE, BEAS-2B or VERO cells were pre-incubated in normocapnia (5% CO2, PCO2 36 mmHg) or hypercapnia (15% CO2, PCO2 108 mmHg), both with normoxia, for 4 days. Expression of ACE2 and sterol regulatory element binding protein 2 (SREPB2), the master regulator of cholesterol synthesis, was assessed by immunoblot or immunofluorescence. Cholesterol was measured in cell lysates by Amplex red assay. Cells cultured in normocapnia or hypercapnia were also infected with Pseudo SARS-CoV-2, a Neon Green reporter baculovirus. For in vivo studies, C57BL/6 mice were exposed to normoxic hypercapnia (10% CO2/21% O2) for 7 days, or air as control, and airway epithelial expression of ACE2, SREBP2, ABCA1, ABCG1 and HMGCS1 was assessed by immunofluorescence. SREBP2 was blocked using the small molecules betulin or AM580, and cellular cholesterol was disrupted using MβCD. Results: Hypercapnia increased expression and activation of SREBP2 and decreased expression of ABC transporters, thereby augmenting epithelial cholesterol levels. Elevated CO2 also augmented ACE2 expression and Pseudo-SARSCoV- 2 entry into epithelial cells in vitro and in vivo. These effects were all reversed by blocking SREBP2 or disrupting cellular cholesterol. Conclusion: Hypercapnia augments cellular cholesterol levels by altering expression of cholesterol biosynthetic enzymes and efflux transporters, leading to increased epithelial expression of ACE2 and entry of Pseudo-SARS-CoV-2 into cells. These findings suggest that ventilatory support to limit hypercapnia or pharmacologic interventions to decrease cellular cholesterol might reduce viral burden and improve clinical outcomes of SARSCoV- 2 infection in advanced COPD and other severe lung diseases.

ROLE of ACTIN REGULATORS in HIV-1, IAV, & SARS-CoV-2 VIRUS-LIKE PARTICLES PRODUCTION

Background: Human immunodeficiency virus (HIV) and Influenza A virus (IAV) remain a global health concern. Further, emergence of novel coronavirus SARS-CoV-2, which rapidly became global pandemic, increases the concern in biomedical research field for antiviral treatment. To develop new antiviral therapy, we must need to understand the molecular and cellular mechanisms involved in assembly and replication. It is known for some viruses (HIV and IAV) that the host actin cytoskeleton has been involved in various stages of the virus life cycle. Regulation of actin cytoskeleton requires several actin binding proteins, which organize the actin filaments (F-actin) into higher order structures such as actin bundles, branches, filopodia and microvilli, for further assistance in viral particle production. Thus, our objective for this work is to understand the role of these actin regulator proteins, like cofilin and one of its cofactor WDR1, in viral particle assembly and release. Methods: Here we used a combination of different experimental methods like RNA interference, immunoblot, immunoprecipitation, immunofluorescence coupled to confocal and STED fluorescence microscopy. In order to study only virus release, and bypass viral entry, we set up a minimal system for virus-like particles production in transfected cells, giving HIV-1 Gag-VLP, Influenza M1-VLP and SARS-CoV-2 MNE-VLP (developed by D. Muriaux lab). For image analysis, we used Image J software. Statistical analysis was performed with non-parametric t-tests or one-way Anova test. Results: Using siRNA strategy, we have shown that upon knock down of actin protein cofilin or WDR1, HIV-1 and IAV particles production increases in contrario to SARS-CoV-2 VLP release. Further, using immunoprecipitation, we report that HIV-1 Gag is able to form an intracellular complex with WDR1 and cofilin. Similarly, IAV-M1, which like HIV Gag-MA binds with plasma membrane phospholipids, is able to form an intracellular complex with cofilin. These results suggested that virus budding from the host cell plasma membrane seemed restricted by the cofilin/WDR1 complex. Finally, using confocal/STED microscopy on cell producing VLP, we observed actin fibers rearrangement with cell protrusions, suggesting a role for actin in viral particles assembly and release. Conclusion: In conclusion, regulators of actin dynamic are involved in HIV-1 Gag, IAV-M1 and SARS-CoV-2 VLP production but play a differential role in assembly and release of these RNA enveloped viruses.

Fibronectin as a Marker of Disease Severity in Critically Ill COVID-19 Patients

The SARS-CoV-2 virus alters the expression of genes for extracellular matrix proteins, including fibronectin. The aim of the study was to establish the relationship between different forms of fibronectin, such as plasma (pFN), cellular (EDA-FN), and proteolytic FN-fragments, and disease severity and mortality of critically ill patients treated in the intensive care unit. The levels of pFN, EDA-FN, and FN-fragments were measured in patients with a viral (N = 43, COVID-19) or bacterial (N = 41, sepsis) infection, using immunoblotting and ELISA. The level of EDA-FN, but not pFN, was related to the treatment outcome and was significantly higher in COVID-19 Non-survivors than in Survivors. Furthermore, EDA-FN levels correlated with APACHE II and SOFA scores. FN-fragments were detected in 95% of COVID-19 samples and the amount was significantly higher in Non-survivors than in Survivors. Interestingly, FN-fragments were present in only 56% of samples from patients with bacterial sepsis, with no significant differences between Non-survivors and Survivors. The new knowledge gained from our research will help to understand the differences in immune response depending on the etiology of the infection. Fibronectin is a potential biomarker that can be used in clinical settings to monitor the condition of COVID-19 patients and predict treatment outcomes.

Blood-Based Biomarkers for COVID-19-Associated Neurological Outcomes

Background: There are no blood screening tests to assess brain molecular alterations linked to neurological alterations in human coronavirus disease 2019 (COVID-19). Evidence indicates long-term brain abnormalities associated with SARS-CoV-2 infection, including cognitive impairment, which may develop into an emerging health problem as many patients are emerging with cognitive abnormalities that may be associated to an increased risk of AD. Promising results from the field of blood-based biomarkers are emerging with the use of extracellular vesicles (EVs). Neuronal-derived EVs (NDEVs) can be isolated from the total pool of EVs in the blood to investigate biomarkers of brain diseases. Methods: Isolation of NDEVs was performed using the ExoQuick ULTRA EV Isolation System, followed by immunoprecipitation with L1CAM antibody. EVs were characterized by nanoparticle tracking analysis, electron microscopy, Exo-Check Array, and ELISA/immunoblotting to detect exosome proteins. Biomarker measurements in the plasma, CSF and EVs from plasma was done by ELISA. A broader analysis of isolated EVs was done by Mass spectrometry. Results: Levels of cytokines were increased in the blood samples from a cohort of 100 COVID-19 patients compared to controls. We had the opportunity to investigate biomarkers in the CSF of 38 patients and observed that the levels of cytokines and biomarkers of neurodegeneration in CSF samples were increased. Conclusions: COVID-19 was associated with increases in CSF and blood cytokines and markers of neurodegeneration. A close follow up in patients that developed COVID-19 symptoms is important to determine the long-term consequences of infection Funding Source: CNPq, FAPERJ, CIHR Keywords: Extracellular Vesicles, Biomarkers, COVID-19

Pharmacologic inhibition of SUMOactivating enzyme potentiates interferon response and T cell-mediated anti-tumor immunity in chronic lymphocytic leukemia (CLL) and lymphoma models

Introduction: CLL is characterized by deficient immunity which clinically manifests as an increased predisposition toward malignancies and infectious complications. T-cells from patients with CLL exhibit a skewed repertoire with a predominance of Tregs as well as impaired immune synapse formation and cytotoxic function. Unlike chemotherapy, novel targeted agents may have beneficial immunomodulatory effects, which may be particularly relevant in the COVID-19 era. Small ubiquitin-like modifier (SUMO) family proteins regulate a variety of cellular processes, including nuclear trafficking, gene transcription, and cell cycle progression, via post-translational modification of target proteins. Sumoylation regulates NFjB signaling, IFN response, and NFAT activation, processes indispensable in immune cell activation. Despite this, the role of sumoylation in T cell biology in the context of cancer is not known. TAK-981 is a small molecule inhibitor of the SUMO-activating enzyme (SAE) that forms a covalent adduct with an activated SUMO protein, thereby preventing its transfer to the SUMO-conjugating enzyme (Ubc9). Here, we investigated the immunomodulatory effects of TAK-981 in CLL. Methods: T cells from patients with CLL were purified using Dynabeads. Activation, proliferation, and apoptosis of CD3+ T cells were studied following T-cell receptor engagement (TCR;aCD3/CD28) with/without 0-1 lM TAK-981. Cytokines were measured after in vitro stimulation. For polarization assays, FACS-sorted naïve CD4+ T cells were cultured for 7 days in control or differentiation media. For gene expression profiling (GEP;Clariom S), RNA was harvested after 3 and 24 h of TCR engagement from FACS-sorted naïve CD4+ T cells. For in vivo immunization experiments, CD4+KJ1-26+ cells were inoculated IV into BALB/cJ mice. Mice received 100 mg IV ovalbumin ± R848 followed by TAK-981 7.5 mg/kg or vehicle control IV twice weekly for 10 days before spleen collection. Both recipient and transplanted splenocytes were analyzed. For analysis of tumor-infiltrating lymphocytes (TILs), BALB/c mice were injected with 1×106 A20 lymphoma cells and treated as above. TAK-981 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA, USA). Results: T cells from patients with CLL demonstrated high baseline protein sumoylation that slightly increased following TCR engagement. Treatment with TAK-981 significantly downregulated SUMO1 and SUMO2/3-modified protein levels, yet did not disrupt early TCR signaling as evidenced by sustained ZAP70, p65/NFjB, and NFAT activation detected by immunoblotting, immunocytochemistry, and GEP. Treatment with TAK-981 resulted in dose-dependent upregulation of the early activation marker CD69 in CD4+ T cells following 72 and 96 h of TCR stimulation vs. control. Meanwhile, the expression of CD25, HLA-DR, and CD40L was delayed in the presence of TAK-981. Interestingly, CD38, an IFN response target, was induced 2-fold in TAK-981-treated cells after 24 h and persisted at high levels at subsequent timepoints. T cell proliferation was reduced in the presence of high (1 lM) but not low/intermediate concentrations of TAK-981, accompanied by reduced S phase entry and decreased synthesis of IL- 2. However, T cells did not undergo apoptosis under those conditions. Targeting SAE in either control or Th1/Treg polarizing conditions facilitated an increase in IFNc and loss of FoxP3 expression (accompanied by decreased IL-2/STAT5), suggesting a shift toward Th1 and away from Treg phenotype, respectively. GEP (Reactome, GSEA) confirmed a dramatically upregulated IFN response in TAK-981-treated CD4+ naïve T cells. Furthermore, targeting SAE enhanced degranulation (CD107a), IFNc, and perforin secretion in cytotoxic CD8+ T cells and potentiated T cell cytotoxicity in allogeneic assays with lymphoma cells (OCI-LY3, U2932) as targets. Consistent with our in vitro data, OVA-stimulated transplanted transgenic KJ1-26+ splenocytes, as well as total CD4+ T cells from recipient mice treated with TAK-981 in vivo exhibited a significant reduction in express on of FoxP3 and an increased production of IFNc. In the A20 syngeneic model, treatment with TAK-981 similarly downregulated FoxP3 expression in CD4+ TILs and induced IFNc secretion in CD8+ TILs. Conclusion: Using a combination of in vitro and in vivo experiments, we demonstrate that pharmacologic targeting of sumoylation with TAK-981 does not impair proximal TCR signaling in T cells obtained from patients with CLL, but leads to rebalancing toward healthy immune T cell subsets via induction of IFN response and downmodulation of Tregs. These data provide a strong rationale for continued investigation of TAK-981 in CLL and lymphoid malignancies.

COVID-19 spike-protein causes cerebrovascular rarefaction and deteriorates cognitive functions in a mouse model of humanized ACE2

COVID-19 pandemic has affected our health and economy. Clinical trials confirmed multiple neurological symptoms due to COVID-19, ranging from headaches, insomnia to stroke, and encephalopathy. More studies are required to unravel the cellular and molecular mechanisms to find a cure for these neurological symptoms. Here, we investigate the effect of COVID-19 spike protein (S-protein) on the cerebrovasculature and cognitive functions in two mouse models that express humanized ACE-2 (h ACE2), a receptor essential for cellular infection and COVID-19 internalization. We hypothesize that COVID-19 S-protein causes cognitive dysfunction via the deterioration of cerebrovascular functions. Methods: S-protein was either injected intravenously or directly into the hippocampus of K-18 (h ACE2 in epithelial cells) or global h-ACE2 knock-in (h ACE2 KI) mice or wild-type mice. Cognitive functions were assessed by Y-maze and Barnes maze. Cerebrovascular density was determined using confocal 3-D image reconstruction. Human brain microvascular endothelial cells (HBMVEC) were treated with S-protein and assessed for apoptosis and inflammatory markers using immunoblotting and RT-PCR. K-18 and h-ACE2 KI mice received intraocular injections of S-protein;retinas were evaluated for vascular cell death and inflammation. Results: S-protein injections caused significant deterioration in memory and learning function of K-18 and h-ACE2 KI mice but not in the wild-type mice (P<0.05). S-protein significantly increased inflammatory mediators, cytokine production, and apoptosis in the brains and HBMVECs (P<0.05). Significant cerebrovascular rarefaction was detected only in K-18 and h-ACE2 KI mice compared to wild-type mice (P<0.05). Retinal vascular cell death and inflammation were significantly increased after S-protein injection. (P<0.05) Conclusions: COVID-19 spike protein decreases cognitive function via increased endothelial cell inflammation, apoptosis, and cerebrovascular rarefaction. Humanized ACE2 animal models are excellent and reliable for investigating the neurological symptoms of COVID-19.

ATN-161 prevents α5 integrin-mediated tight junction disruption caused by SARS-CoV-2 and its delta variant in brain endothelial cells

Patients with significant cerebrovascular comorbidities (e.g. brain ischemia, vascular dementia) are more affected and are more likely to have worsened post-acute neurologic sequelae after SARSCoV-2 infection. This may be due to viral invasion and propagation in brain endothelial cells (BECs) and disruption of the blood-brain barrier (BBB). Viral spike protein used to bind and infect host cells encodes an arginine-glycine-aspartic acid (RGD) motif that it may use to bind integrins cell receptors that play an important role in cerebrovascular integrity. Therefore, integrins may represent an acute and post-acute SARS-CoV-2 therapeutic target. However, the interplay between vascular dysregulation, integrin function, and COVID-19 is unclear. As we have previously demonstrated that activation of the integrin α5 plays a key role in BBB breakdown, stroke injury, OGD/R, SARS-CoV-2 infection, and its inhibition with the clinically validated peptide ATN-161 is therapeutic in these conditions, we hypothesize that SARS-CoV-2 alters BEC α5 integrin (and associated tight junction protein) expression as a means of infecting and altering cerebrovascular integrity, and this can be prevented by ATN-161. Methods: Mouse BECs (bEnd3) were inoculated with heat-inactivated SARS-CoV-2 (Isolate USAWA1/2020) or delta variant of SARS-CoV-2 spike protein for 24 h then later exposed to hypoxia for 6h to model the effects of in vivo pulmonary infection. Cells were pretreated with ATN-161 (1, 5, and 10μM) 1h before SARS-CoV-2 challenge and during hypoxia. α5 and claudin-5 proteins were analyzed by immunoblotting. Results: Both SARS-CoV-2 inoculations induced integrin α5 and decreased claudin-5 expression (delta > SARS-CoV-2) in a dose-dependent fashion, although higher doses of SARS-CoV-2 (2.5 and 5 μg) had no effect on these proteins. SARS-CoV-2 spike protein challenge at 0.5 μg followed by hypoxia resulted in increased α5 and decreased claudin-5 expression in either hypoxia or SARSCoV-2+hypoxia combination. ATN-161 (10μM) pretreatment inhibited SARS-CoV-2+hypoxia-induced α5 upregulation and restored claudin-5 loss. In addition to its demonstrated anti-viral effects, ATN161 may be an important therapy for SARS-CoV-2-mediated cerebrovascular injury.