SUBPOPULATION COMPOSITION OF PERIPHERAL BLOOD LYMPHOCYTES IN COVID-19.

The pathogenesis of severe coronavirus infection COVID-19 is associated with activation of immune system, cytokine storm, impaired blood clotting, microvascular thrombosis, organ ischemia and multiple organ dysfunction syndrome. The role of various lymphocyte subpopulations in COVID-19 is still debated. The aim of our study was to analyze the subpopulational profile of peripheral blood lymphocytes in COVID-19 patients as compared with healthy donors. The study included 20 COVID-19 patients (11 males and 9 females,) and 26 healthy donors. Average age of the patients was 52 and 56 years, respectively. Clinical examinations were performed by standard laboratory methods. Peripheral blood lymphocytes were isolated in the Ficoll gradient. The cells were stained with antibodies to specific antigens of main lymphocyte populations, endothelial cells, and apoptotic cell markers. The analysis was performed by flow cytometry. The results showed that all patients had elevated C-reactive protein (14- to 35-fold), ferritin (1.2- to 13-fold), D-dimers (1.2- to 90-fold). 55% of men had a decrease in the absolute number of lymphocytes, in women this index was at the low normal limit. Cytometric analysis showed that, among peripheral blood lymphocytes, the proportion of functional cells expressing the CD45 marker ranged from 2 to 12% in 70% of patients, as compared with 80-99% among the donors. The proportion of CD45+ lymphocytes significantly correlated with the level of hemoglobin, but not with the levels of inflammatory biochemical markers. Among the functional lymphocytes of patients, there was a decrease in the proportion of CD3+, CD4+, CD8+T cells, increased proportion of natural killer CD56+ and the apoptotic (AnnexinV+) cell contents, but the proportion of CD19 and HLA-DR+B cells was not changed. Analysis of the lymphocyte (LC) subpopulations that did not express CD45 marker showed that this fraction contained different lymphocyte subsets with reduced expression of CD4, CD8, CD19, CD56 etc. in the blood of patients and donors. Higher percentage of endothelial cells expressing CD62P marker made the difference between patients and donors. Laboratory determination of lymphocyte subsets in blood samples of COVID-19 patients does not reflect the real severity pattern of the disease, thus requiring studies of the CD45-expressing functional cell populations.Copyright © Svirshchevskaya E.V. et al., 2023 The article can be used under the Creative Commons Attribution 4.0 License.

SUBPOPULATION COMPOSITION OF PERIPHERAL BLOOD LYMPHOCYTES IN COVID-19.

The pathogenesis of severe coronavirus infection COVID-19 is associated with activation of immune system, cytokine storm, impaired blood clotting, microvascular thrombosis, organ ischemia and multiple organ dysfunction syndrome. The role of various lymphocyte subpopulations in COVID-19 is still debated. The aim of our study was to analyze the subpopulational profile of peripheral blood lymphocytes in COVID-19 patients as compared with healthy donors. The study included 20 COVID-19 patients (11 males and 9 females,) and 26 healthy donors. Average age of the patients was 52 and 56 years, respectively. Clinical examinations were performed by standard laboratory methods. Peripheral blood lymphocytes were isolated in the Ficoll gradient. The cells were stained with antibodies to specific antigens of main lymphocyte populations, endothelial cells, and apoptotic cell markers. The analysis was performed by flow cytometry. The results showed that all patients had elevated C-reactive protein (14- to 35-fold), ferritin (1.2- to 13-fold), D-dimers (1.2- to 90-fold). 55% of men had a decrease in the absolute number of lymphocytes, in women this index was at the low normal limit. Cytometric analysis showed that, among peripheral blood lymphocytes, the proportion of functional cells expressing the CD45 marker ranged from 2 to 12% in 70% of patients, as compared with 80-99% among the donors. The proportion of CD45+ lymphocytes significantly correlated with the level of hemoglobin, but not with the levels of inflammatory biochemical markers. Among the functional lymphocytes of patients, there was a decrease in the proportion of CD3+, CD4+, CD8+T cells, increased proportion of natural killer CD56+ and the apoptotic (AnnexinV+) cell contents, but the proportion of CD19 and HLA-DR+B cells was not changed. Analysis of the lymphocyte (LC) subpopulations that did not express CD45 marker showed that this fraction contained different lymphocyte subsets with reduced expression of CD4, CD8, CD19, CD56 etc. in the blood of patients and donors. Higher percentage of endothelial cells expressing CD62P marker made the difference between patients and donors. Laboratory determination of lymphocyte subsets in blood samples of COVID-19 patients does not reflect the real severity pattern of the disease, thus requiring studies of the CD45-expressing functional cell populations.Copyright © Svirshchevskaya E.V. et al., 2023 The article can be used under the Creative Commons Attribution 4.0 License.

SYSTEMIC OZONE THERAPY AND ITS IMMUNOMODULATORY EFFECTS IN COVID-19 PATIENTS

Objective: To evaluate the effect of systemic ozone therapy (OT) on the concentration of pro-inflammatory and anti-inflammatory cytokines in the blood in the complex treatment of COVID-19 patients. Material(s) and Method(s): The study included 65 patients with a confirmed diagnosis COVID-19 characterized by a moderate and severe course of the disease. The patients were admitted to the Infectious Disease Hospital of the V.I. Kulakov National Medical Research Centre for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia. The patients' age ranged from 29 to 78 years. All patients were treated in accordance with the Temporary Guidelines of the Ministry of Health of Russia "Prevention, Diagnosis and Treatment of Coronavirus Infection (COVID-19)". Two groups of patients were randomly formed. The first group included 35 patients whose complex therapy included OT: intravenous administration of 400 ml of ozonated saline solution with an ozone concentration of 4 mg/L;a total course consisting of 6 procedures performed every other day. The second group included 30 patients who did not have OT. Clinical and laboratory parameters were evaluated on admission to the Infectious Disease Hospital and after two weeks of complex treatment;clinical, laboratory, special, statistical research methods were used. The content of cytokines GM-CSF, IFN-gamma, TNF-alpha, IL-2, IL-4, IL-6, IL-8, IL-10 and their ratios were determined with a multiplex method on the Bioplex 200 analyzer (Bio-Rad, USA) using Bio-Plex Pro Human Cytokine 8-plex Panel (Bio-Rad, USA). Result(s): On admission to the Infectious Disease Hospital 47/65 (72,3%) patients had a moderate course of the disease and 18/65 (27.7%) patients had a severe one. The length of hospital stay in the group of patients with OT averaged 12.2 (2.7) (8-17) days, and it was 17.9 (4.2) (12-26) days in the group of patients who did not have OT. On admission, all patients had an increase in the level of C-reactive protein in their blood serum;the cytokine content in patients of the groups differed from the initial cytokine level and it was different between groups after two weeks of therapy. The medians of the pro-inflammatory cytokines IL-2, IL-6, IL-8 were particularly different. The content of these cytokines remained elevated in the second group of patients who did not have OT, compared with the baseline data and compared with the first group. The study of anti-inflammatory cytokines showed that the patients of the first group who had OT demonstrated a significantly higher IL-10 content compared to IL-10 content in the patients of the second group. Ratios of pro-and anti-inflammatory cytokines IL-2/IL-10, IL-2/ IL-4, IL-6/IL-10, IL-6/IL-4, IL-8/IL-10, IL-8/IL-4, TNF-alpha/IL-4 in the first group of patients with OT significantly decreased compared to the baseline indicator, which can be suggestive of a marked decrease in the activity of the inflammatory process. Conclusion(s): The positive effect of systemic OT on the clinical course of the disease has been revealed. Laboratory indicators in COVID-19 patients have shown a decrease in the severity of the inflammatory response. Besides having anti-inflammatory and immunomodulatory effect, OT helps to stop the process, improve the condition of patients and reduce the length of hospital stay. Systemic OT should be considered as an additional adjuvant method in the complex treatment of patients with SARS-CoV-2 infection. Copyright © A group of authors, 2022.

Myocardial infarction versus COVID-19 blood secretome

Background: Both myocardial infarction (MI) and COVID-19 are characterized by cytokine storm in blood. Purpose: The objective of this study was to compare the concentration of 39 cytokines, chemokines, and growth factors in blood sera of patients with MI, COVID-19 (COV), and healthy donors. Methods: Patients' blood was collected within 1-2 days after hospitalization in the cardiovascular or COVID intensive care units. All COV patients were in a severe condition;all had increased C reactive protein, 86 and 95% had increased ferritin and D-dimers levels accordingly, 8-10 times decreased lymphocyte numbers. The analysis of the humoral factors in blood serum of MI (n=22), COV (n=23) and donors (n=27) was performed using a 39-plex cytometric analysis. Results: Among all factors analyzed TGFa, IL-1b, 2, 3, 5, 9, 13, 17A were almost not detectable both in patient and donor sera. The concentrations of the other 31 humoral factors in normal sera differed significantly from 0 to 22000 pg/mL. We divided them into house-keeping factors HKF ranged from 1000 to 22000 pg/mL;sentinel innate immunity factors SIF (30-200 pg/mL), and acute phase factors APF (0-30 pg/mL). HKF were detected in all samples. Among SIF and APF IL-1a, G-CSF, IFNa2, IL-7, MIP-1a, IL- 12, and IFNg were detected in 56-80% donor blood while IL-1RA, MCP-3, IL-2, 6, 10, 12, 15, FLT-3F, GM-CSF, TNF-b - only in 10-55%. At the same time all MI patients were 100% positive in all these factors showing extensive activation of blood secretome. Among low incidence APF cytokines in COV patients, percentage of IL-1RA, MCP-3, IFNa2, IL-6, 10, 15, FLT-3L negative sera decreased 3-5 times;and all sera were positive for MIP-1a and IL-12. At the same time TNF-a level decreased significantly from 0 in control to 85% of negative sera in COV patients. Summarized results are shown as the ratios of factor concentrations in MI or COV sera to normal control (Fig). Blood secretome of MI changed more significantly than of COV patients. The major factors (shown in red) in MI were IL-6, IL-12, IFNg, FLT-3L, GM-CSF, and IL-15, which increased 12, 9, 6, 6, 6, and 5 times accordingly. In COV sera IL-6, IL-10, IP-10, and MCP-3 increased by 28, 12, 10, and 9 times accordingly. Less expressed however significant increases are marked with asterisks. Conclusions: Acute MI is characterized by severe disturbances in blood secretome with an increased level of 25 out of 39 factors studied. Contrary to it, in COV patients the levels of IL-6, 10, IP-10, and MCP-3 were more enhanced while only 15 out of 31 exceeded normal levels.

Immune status of covid-19 patients with different disease severity

Aim. To investigate the immune status and compare immunological parameters in COVID-19 patients with different disease severity. Materials and methods. The prospective study included 62 patients with COVID-19. The patients were stratified into three groups based on the disease severity, including mild (group 1, n=29), moderate (group 2, n=17), and severe (group 3, n=16) forms of COVID-19. On days 3–7 from the onset of the disease, peripheral blood lymphocytes were phenotyped by flow cytometry. Cytokine concentrations were measured using a multiplex immunoassay-standard 48-plex Bio-Plex Pro™ Human Cytokine Screening test system (Bio-Rad, USA) on a flow-based laser immuno-analyzer Bio-Plex 200. Results. Patients with severe COVID-19 had higher levels of leukocytes, neutrophils, CRP, and lower relative and absolute lymphocyte counts. There were low counts of CD3+, CD3+CD4+, CD3+CD8+, and T-lymphocytes expressing the activation marker HLA-DR (CD3+HLA-DR+), NK-cells, and PAN. In group 3, changes in 39 of the 48 investigated soluble factors were observed. Conclusion. High levels of leukocytes, neutrophils, CRP, neutrophilic-leukocyte index, low levels of absolute and relative lymphocyte counts, pronounced changes in immunological parameters, a systemic inflammatory reaction associated with the release of mediators called cytokines ("cytokine storm") predispose to a severe course of COVID-19. © A group of authors, 2021.

Results of multicenter monitoring of hemostasis parameters in patients with COVID-19

Materials and methods: a prospective non-randomized pilot multicenter study of the informativeness and clinical significance of hemostasis laboratory tests in 1210 patients with COVID-19 in disease course, including favorable and unfavorable outcomes, was performed. Hemostasis was assessed using clotting tests and D-dimer concentration, thromboelastography (TEG) and thrombodynamics (TD). Results: comparison of COVID-19 laboratory parameters and clinical picture showed that 75% of patients have pronounced activation of the plasma coagulation system upon admission to the hospital. Hypercoagulation is recorded in all tests, reaching a maximum in patients with subtotal (CT-3) and total (CT-4) lung lesion and/or resuscitation patients with a clinical picture of pulmonary embolism and unfavorable outcome. Low molecular weight heparins (LMWH) in a standard dosage leads to suppression of the initial hypercoagulable syndrome in more than half of the patients (from 75 to 31%). All patients without LMWH laboratory effect developed thrombotic complications. For clotting tests, insufficient sensitivity to changes in hemostasis against the background of LMWH was revealed. The D-dimer test effectively correlates with the severity and outcomes of COVID-19, but is not suitable for the control of heparin therapy, which is associated with the effect of lysis of existing blood clots and the lack of response to a decrease in the coagulation activity of patients. Methods of thromboelastography and thrombodynamics effectively record a decrease in the activity of the coagulation system and can be used to control heparin therapy. The correlation coefficient between the methods was 0,77. The dynamic indices of D-dimers, TEG and TD in severe patients and, especially, in patients with fatal outcomes revealed the greatest sensitivity to the control of heparin therapy in the Thrombodynamics test, which allows, along with thrombosis, to record hypercoagulable states and the risk of bleeding, which are the outcome of thrombohemorrhagic syndrome in patients with COVID-19. Материалы и методы исследования: проведено проспективное открытое нерандомизированное пилотное многоцентровое исследование информативности и клинической значимости лабораторных тестов гемостаза у 1210 пациентов с COVID-19 в динамике заболевания, включая благоприятные и неблагоприятные исходы. Оценку гемостаза проводили с использованием клоттинговых тестов и концентрации D-димера, тромбоэластографии (ТЭГ) и тромбодинамики (ТД). Результаты: при сопоставлении лабораторных показателей и клинической картины COVID-19 показано, что у 75% больных при поступлении в стационар наблюдается выраженная активация плазменной системы свертывания. Гиперкоагуляция фиксируется по всем тестам, достигая максимума у больных с субтотальным (КТ-3) и тотальным (КТ-4) поражением легких и/или реанимационных больных с клинической картиной тромбоэмболии легочной артерии и неблагоприятным исходом. Назначение низкомолекулярных гепаринов (НМГ) в стандартной дозировке приводит к подавлению исходного гиперкоагуляционного синдрома более чем у половины больных (с 75 до 31%). Все пациенты без лабораторного эффекта НМГ развили тромботические осложнения. Для клоттинговых тестов выявлена недостаточная чувствительность к изменениям гемостаза на фоне НМГ. Тест на D-димер эффективно коррелирует с тяжестью и исходами COVID-19, но непригоден для контроля гепаринотерапии, что связано с эффектом лизиса существующих тромбов и отсутствием ответа на снижение коагуляционной активности больных. Методы ТЭГ и ТД эффективно регистрируют снижение активности свертывающей системы и могут использоваться для контроля гепаринотерапии. Коэффициент корреляции между методами составил 0,77. Заключение: динамические индексы D-димеров, ТЭГ и ТД у тяжелых больных и особенно у пациентов с летальными исходами выявили наибольшую чувствительность к контролю гепаринотерапии у теста ТД, что позволяет наряду с тромбозами фиксировать гиперкоагуляционные состояния и риск кровотечений - исходы тромбогеморрагического синдрома у больных с COVID-19.

Plasma hemostatic system in patients with the novel coronavirus disease 2019 (COVID-19)

Aim. To investigate plasma hemostasis in patients with different levels of COVID-19 severity. Materials and methods. The study included 46 patients with confirmed COVID-19, who were stratified into four groups based on disease severity as mild (1), moderate (2), severe (3), and critically severe (4). Laboratory investigations included APTT, Quick's prothrombin time, concentration of D-dimer, fibrinogen, ATIII, and platelet count. Results. D-dimer concentration >450 ng/ml was found in 100% of patients increasing from 973 (545–1635) ng/ml in group 1 to 13513 (7627–22512) ng/ml in group 4 (p<0.05). Patients in group 2 had the highest fibrinogen level averaging 6.1(3.9–6.4) g/L. The highest and lowest platelet counts were in groups 1 (323x109) and 4 (155×109), respectively, (p <0.05). The ATIII levels in groups 1 and 2 were within the normal range, while in groups 3 and 4, they were decreased to 76% and 64%, respectively (p <0.05). All patients enrolled in the study had normal values of APTT and Quick's prothrombin time. Unfavorable outcomes were observed at a D-dimer concentration> 3633 ng/ml (specificity 92.3%, sensitivity 100%, AUC 0.94, p <0.001, positive predictive value 99.1%, and negative predictive value 100%) and ATIII <70.8% (specificity 70%, sensitivity 82.6%, AUC 0.85, p <0.001, positive predictive value 30.5%, and negative predictive value 96.1%). Conclusion. The levels of D-dimer, fibrinogen, and ATIII were found to be associated with the severity of COVID-19.