Serum vitamin D content in children with SARS-CoV-2 infection.

Aim of study. To study the total level of 25-hydroxyvitamin D in children with SARS-CoV-2 infection (COVID-19). Material and Methods. A total of 82 children aged 0-17 diagnosed with SARS-CoV-2 infection were enrolled. Depending on the severity of clinical symptoms, all children were divided into three groups according to the COVID-19 severity: asymptomatic, mild and moderate. The serum level of vitamin D in all patients was tested via the immunochemical method. Results. It was found that children with SARS-CoV-2 infection had lower serum level of vitamin D (29.92 [22.22;28.07] ng/ml) as compared with the control group (36.43 [32.05;44.08] ng/ml;p<0.001). A total of 90% of the children with SARS-CoV-2 infection were diagnosed with insufficiency or deficiency of vitamin D (<30 ng/ml). Only 10 % of the patients had normal levels of vitamin D. The insufficiency of vitamin D was found more often amongst children aged 0-11 and deficiency of total 25-hydroxyvitamin D was more common for children aged 12-17. The difference in serum levels of vitamin D depending on the severity of SARS-CoV-2 infection was not found. Male children with SARS-CoV-2 infection showed lower levels of vitamin D (p=0.013). Conclusion. A total of 90 % of the children with SARS-CoV-2 infection had insufficiency or deficiency of vitamin D regardless of the severity of clinical symptoms.Copyright © 2021, Krasnoyarsk State Medical University. All rights reserved.

Immunosuppressive therapy after organ transplantation - challenges of treatment during the COVID-19 pandemic

Organ transplantation in the final stages of chronic disease or in case of acute failure is an accepted procedure of treating patients that has been developing for many years. After the organ transplantation procedure, immunosuppressive therapy is started, which strikes a balance between the modulation of the immune system in order to avoid organ rejection or the harmful effects of immunosuppression. Commonly used immunosuppressants for the treatment of transplant patients are from the group of calcineurin inhibitors (cyclosporine, tacrolimus) and mTOR inhibitors (sirolimus, everolimus). Mycophenolic acid, leflunomide and glucocorticoids are used as supportive therapy, and with the discovery of biological therapy, therapeutic monoclonal antibodies directed against various cellular targets have been developed. Optimization and laboratory monitoring of immunosuppressive concentrations is necessary after transplantation in order to avoid graft rejection and the occurrence of unwanted side effects. The most commonly used methods for therapeutic drug monitoring in clinical laboratories are immunochemical methods characterized by high levels of automation but also have major shortcomings such as insufficient specificity and standardization, which is why the method of choice for therapeutic monitoring is liquid chromatography-tandem mass spectrometry whose main characteristic is specificity and selectivity. Therefore, when measuring the concentration of immunosuppressants, it is important to state the method of determination. The global spread of Coronavirus disease (COVID-19) has affected organ donation and transplantation and is actively trying to clarify the role of immunosuppressive therapy in the disease process because it is extremely difficult to strike a balance between suppressing the immune response to prevent organ rejection and control inflammation during COVID-19 disease. Given the complexity of treating the transplant population of patients with COVID-19, there is a clear need for a systemic approach to treatment, which will consequently lead to better outcomes.

Eicosapentaenoic acid favorably modulates protein expression in pulmonary endothelial inflammation

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

Single cell profiling in COVID-19 associated acute kidney injury reveals patterns of tubule injury and repair in human

Background: Acute Kidney Injury (AKI) affects up to one in two critically ill patients. The cellular mechanisms of kidney tubule repair after acute kidney injury are poorly characterized in humans. Methods: We recruited 5 patients admitted to the Geneva University Hospital's Intensive Care Unit for severe COVID19 and experiencing AKI. For each of them, a kidney biopsy was performed before the planned withdrawal of resuscitation measures. We further applied single-cell RNA sequencing to analyze the kidney in the first days after acute injury. Results: After data processing and quality control, we obtained 20,165 single-cell transcriptomes. The most prominent finding in the snRNAseq analyses was in the proximal tubule (PT) compartment. We defined two cell populations corresponding to mature and undifferentiated PT cells, connected by two cell state transitions (Figure 1). Undifferentiated PT cells display an injured pattern characterized by metabolic impairment, reduction of the tubule transport function, and expression of injury markers confirmed in immunochemistry. We found that tubule repair follows two converging patterns involving the plasticity of mature tubule cells and the expansion and differentiation of progenitor-like cells. Tubule repair by cell plasticity displayed substantial similarities among mice and men and determined the transient expansion of undifferentiated tubule cells with altered functional and metabolic properties. Progenitorlike cells marked by PROM1 proliferated in response to injury and followed a differentiation process characterized by the sequential activation of the WNT, NOTCH, and HIPPO signaling pathways. Conclusions: Here we generated the first map of PT injury and repair in humans. Taken together, our analyses reveal cell states transitions and fundamental cellular hierarchies underlying kidney injury and repair in patients.

Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein.

The current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus and is required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, seven VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange-Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta, and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs.

SARS-CoV-2 infection serology: a useful tool to overcome lockdown?

The outbreak of 2019 novel coronavirus disease (Covid-19) caused by SARS-CoV-2 has spread rapidly, inducing a progressive growth in infected patients number. Social isolation (lockdown) has been assessed to prevent and control virus diffusion, leading to a worldwide financial and political crisis. Currently, SARS-CoV-2 RNA detection in nasopharyngeal swab takes place by real-time PCR (RT-qPCR). However, molecular tests can give some false-negative results. In this context, serological assays can be useful to detect IgG/IgM antibodies, to assess the degree of immunization, to trace the contacts, and to support the decision to re-admit people at work. A lot of serological diagnostic kits have been proposed on the market but validation studies have not been published for many of them. The aim of our work was to compare and to evaluate different assays analytical performances (two different immunochromatographic cards, an immunofluorescence chromatographic card, and a chemiluminescence-automated immunoassay) on 43 positive samples with RT-qPCR-confirmed SARS-CoV-2 infection and 40 negative control subjects. Our data display excellent IgG/IgM specificities for all the immunocromatographic card tests (100% IgG and 100% IgM) and for the chemiluminescence-automated assay (100% IgG and 94% IgM); IgG/IgM sensitivities are moderately lower for all methods, probably due to the assay viral antigen's nature and/or to the detection time of nasopharyngeal swab RT-qPCR, with respect to symptoms onset. Given that sensitivities (around 94% and 84% for IgG and IgM, respectively) implicate false-negative cases and given the lack of effective vaccines or treatments, the only currently available procedure to reduce SARS-CoV-2 transmission is to identify and isolate persons who are contagious. For this reason, we would like to submit a flowchart in which serological tests, integrated with nasopharyngeal swab RT-qPCR, are included to help social and work activities implementation after the pandemic acute phase and to overcome lockdown.