Coronavirus disease (COVID-19) has become a global pandemic. COVID-19 patients need immediate diagnosis and rehabilitation, which makes it urgent to identify new protein markers for a prognosis of the severity and outcome of the disease. The aim of this study was to analyze the levels of interleukin-6 (IL-6) and secretory phospholipase (sPLA2) in the blood of patients regarding the severity and outcome of COVID-19 infection. The study included clinical and biochemical data obtained from 158 patients with COVID-19 treated at St. Petersburg City Hospital No. 40. A detailed clinical blood test was performed on all patients, as well as an assessment of IL-6, sPLA2, aspartate aminotransferase (AST), total protein, albumin, lactate dehydrogenase (LDH), APTT, fibrinogen, procalcitonin, D-dimer, C-reactive protein (CRB), ferritin, and glomerular filtration rate (GFR) levels. It was found that the levels of PLA2, IL-6, APTV, AST, CRP, LDH, IL-6, D-dimer, and ferritin, as well as the number of neutrophils, significantly increased in patients with mild to severe COVID-19 infections. The levels of IL-6 were positively correlated with APTT; the levels of AST, LDH, CRP, D-dimer, and ferritin; and the number of neutrophils. The increase in the level of sPLA2 was positively correlated with the levels of CRP, LDH, D-dimer, and ferritin, the number of neutrophils, and APTT, and negatively correlated with the levels of GFR and lymphocytes. High levels of IL-6 and PLA2 significantly increase the risk of a severe course by 13.7 and 2.24 times, and increase the risk of death from COVID-19 infection by 14.82 and 5.32 times, respectively. We have shown that the blood levels of sPLA2 and IL-6 increase in cases which eventually result in death and when patients are transferred to the ICU (as the severity of COVID-19 infection increases), showing that IL-6 and sPLA2 can be considered as early predictors of aggravation of COVID-19 infections.
We studied the differences in the characteristics of T-cell immunity in clinically healthy volunteers of three groups: “no previous COVID-19, not vaccinated”, “recovered”, and “vaccinated” as well as the relationship between the presence of IFNγ-releasing T cells in response to stimulation with peptide pools overlapping the main S, N, M, ORF3, and ORF7 protein sequences and the presence of IgG to the SARS-CoV-2 S protein. In the “no previous COVID-19, non-vaccinated” group, T cells specific to both S protein and other virus proteins were absent in 95% subjects. In the “recovered from COVID-19” group, T cells specific to the spike protein were present in samples from 39% subjects. In the same group, T-cell immunity to other viral proteins was present in 58% subjects. In vaccinated subjects, specific T cells responding to stimulation with S protein peptides were found in 47% cases and Т cells specific to N, M, ORF3, ORF7 proteins were detected in only 22% subjects.
Secretory phospholipases A2 (sPLA2) represent a large superfamily of enzymes with a molecular weight of 14-19 kDa, including 15 groups and more than 30 isoforms belonging to four types: secretory (sPLA2), cytosolic (cPLA2), calcium-independent (iPLA2) and lipoprotein-associated phospholipase A2 (LP-PLA2, PAF-AH). Eleven species of secretory sPLA2s (IB, IIA, IIC, IID, IIE, IIF, III, V, X, XIIA, and XIIB) have been found in mammals, performing versatile functions and participating in the pathogenesis of a wide range of diseases. On the one hand, sPLA2 may promote elimination of damaged, apoptotic cells by hydrolyzing membrane phospholipids, and exerts a strong bactericidal and antiviral properties, including pronounced effects against antibiotic-resistant strains of microorganisms. In this regard, the use of sPLA2 may represent a new strategy for the treatment of bacterial and viral infections. Moreover, due to the action of sPLA2 on its substrates, a number of biologically active molecules (arachidonic, lysophosphatidic acids, lysophospholipids, fatty acids, prostaglandins, leukotrienes, thromboxanes) are formed, which provide strong inflammatory, detergent, coagulating effects and increase vascular permeability. This pro-inflammatory role of sPLA2 may explain its increase levels and activity in cardiovascular, respiratory, autoimmune, metabolic, oncological, bacterial and viral disorders. The review article presents a classification of sPLA2 isoforms, their substrates, regulatory factors, biological significance, and mechanisms of their strong bactericidal, virucidal, and pro-inflammatory activity in the heart and lung disorders, autoimmune, metabolic, bacterial, and viral diseases. In particular, the mechanisms of the selective action of sPLA2 against Gram-positive and Gram-negative microorganisms are discussed. We consider diagnostic and prognostic significance, correlations between elevated levels and activity of sPLA2 and distinct clinical symptoms, severity and outcome in the patients with coronary heart disease (CAD), acute myocardial infarction (AMI), atherosclerosis, acute inflammatory lung injury (ALI), respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, bronchial asthma, bacterial infections, septicemia and viral (COVID-19) infections. The opportunity of using sPLA2 as a biomarker of the severity and outcome of patients with chronic obstructive pulmonary disease, bacterial infections, sepsis and viral infections, including COVID-19, is also considered.
РезюмеВведение. Эпидемия COVID-19 -крупнейшая пандемия в современной истории. Согласно данным Всемирной организации здравоохранения, на сегодняшний день официально зарегистрировано более 670 млн случаев заражения COVID-19, но, по мнению специалистов, реальное количество случаев COVID-19 значительно больше. Всеохватность пандемии вызвала необходимость самой массовой вакцинации в истории, но даже после начала кампании по вакцинации распространение заболевания радикально не уменьшилось. Всеобщая иммунизация популяции была выбрана в качестве одной из стратегий борьбы с пандемией. Одновременно с этим в научной литературе появляются противоречивые данные о влиянии специфического иммунитета на течение и исход заболевания.Цель настоящего исследования -выяснить взаимосвязь между наличием специфического Т-клеточного и/или гуморального иммунного ответа на SARS-CoV-2 у COVIDнаивных пациентов и исходом заболевания.Материал и методы. Используя метод ELISpot, изучали наличие клеточного иммунитета к наиболее важным мишеням (S-, N-, M-белки) SARS-CoV-2 (дельта-вариант) у ранее наивных к вирусу пациентов (n = 97). С помощью мультиплексного анализа было проведено качественное определение IgG к различным эпитопам SARS-CoV-2. Выделена подгруппа с более строгими критериями включения, чтобы минимизировать влияние коморбидности и возраста пациента на исход заболевания (n = 40).Результаты. На 8-й день заболевания у 100 % пациентов отсутствовал Т-клеточный иммунитет. Его формирование завершалось к 17-му дню заболевания. Выживаемость пациентов не зависела от наличия специфического гуморального и клеточного иммунитета против SARS-CoV-2. Отсутствие коморбидности (ожирение, диабет, нейродегенеративные заболевания, онкология) и молодой возраст пациента значимо не сказывались на результате.Заключение. На основании результатов проведенного исследования нами не обнаружено достоверной зависимости между наличием гуморального или клеточного иммунитета и исходом заболевания у не иммунизированных ранее пациентов, как в общей группе пациентов, так и в группе пациентов с дополнительными критериями включения.
Aim. To optimize the technique for the isolation and storage of ribonucleic acid (RNA) from whole blood and leukocyte fraction.Materials and methods. Comparison of isolation quality was carried out for RNA samples obtained from 228 leukocyte samples and 198 whole blood samples. Isolation was performed from fresh and frozen samples using ExtractRNA™ reagent and a MagNA Pure Compact automated system. Various methods of removing erythrocytes (centrifugation and treatment with hemolytic agents from two manufacturers) were tested, as well as freezing with and without preservatives for subsequent RNA isolation.Results. Twenty-one combinations of conditions were tested. The highest quality RNA was isolated by manual extraction using the ExtractRNA™ reagent from a fresh leukocyte fraction, purified by the Amplisens hemolytic agent (successful extraction — 94%, median RIN=8,4); frozen in IntactRNA™, purified by leukocyte fraction centrifugation (successful extraction — 100%, median RIN=8); frozen in ExtractRNA™, purified by leukocyte fraction centrifugation (successful extraction — 100%, median RIN=9,3).Conclusion. RNA can be isolated from frozen blood fractions, which is not inferior in quality to that isolated from fresh samples. Thus, it is not necessary to isolate RNA immediately after the receipt of biological material.
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