Impaired adaptive immune response in COVID-19 convalescent patients with hematological malignancies.

OBJECTIVES: The immune dysregulation during SARS-CoV-2 has the potential to worsen immune homeostasis after recovery. Patients with hematological malignancies with COVID-19 have changes both in the innate and adaptive immune responses. Little is known about the severity of immune dysfunction following recovery from COVID-19 in hematological patients. METHODS: Here, we performed a comprehensive analysis of the lymphocyte subsets in peripheral blood mononuclear cells by FACS Canto II in 55 patients, including 42 with hematological malignancies 4-6 weeks after COVID-19. RESULTS: Hematological COVID-19 convalescents had deep reduction in CD3+ T cells, including helper T cells (CD3 + CD4+), naïve helper T cells (CD3 + CD4 + CD45RA+), and memory CD4+ T cells among with extremely low levels of Treg cells and decreased expression of both TCRα/ß and TCRγ/δ. Severe immune dysregulation in hematological convalescents was expressed by increased activation of T lymphocytes, both as elevated levels of activated T cells (CD3 + HLA-DR+) and activated cytotoxic T cells (CD3 + CD8 + HLA-DR+). CONCLUSIONS: Our findings showed a profound impairment of the adaptive immune response in hematological convalescents which might be a result of persistent activation of T cells. Convalescents with lymphoid malignancies showed more pronounced depletion of key T lymphocytes subpopulations in creating an effective adaptive response and immune memory.

Features of respiratory tests (hydrogen + methane), indirect liver elastometry data in COVID-19 convalescents (pilot study)

Purpose of the work: to study liver elasticity indicators, hydrogen and methane levels in exhaled air, and their associations with clinical and biochemical parameters for patients who underwent COVID-19. Materials and methods. We examined 30 patients (mean age 51.8±2.91) who underwent COVID-19 (confirmed by SARSCoV-2 RNA test or SARS-CoV-2 antigen) 12-16 weeks after the onset of the first symptoms, of which 11 were diagnosed with pneumonia. 19 people (mean age 47.1±3.09) who did not have COVID-19 made up the comparison group. The patients underwent a clinical and biochemical study, the degree of liver fibrosis was determined (FibroScan® 502 Echosens, France), the levels of hydrogen (H2) and methane (CH4) in the exhaled air were measured (baseline and after taking lactulose solution) (GastroCheck Gastrolyzer, Bedfont Scientific Ltd., England). Results. Past COVID-19 infection was directly correlated with age (r=0.331, p=0.022), male gender (r=0.324, p=0.025), and presence of liver fibrosis (r=0.291, p=0.044). COVID-19 survivors were more likely to have liver fibrosis (p<0.001) and higher liver elasticity in kPa (p=0.018) with overweight and obesity (63.3%) and elevated body mass index (p= 0.03) compared with the control group. The presence of liver fibrosis was associated with moderate pneumonia (p<0.001). Among those who had COVID-19, there were significantly more non-producers of methane (p=0.02), fewer people with an average level of methane in exhaled air (p=0.016). In COVID-19 convalescents, bacterial overgrowth syndrome (BOS) was detected less frequently than in controls (p=0.04), but signs of delayed intestinal transit were more often recorded (p<0.05). The presence of liver fibrosis in survivors of COVID-19 is associated with BOS detection (23.3% vs. 5.2%, p<0.001), which probably contributes to the pathogenesis of liver damage. Hydrogen levels at 120 min and methane at 60 min after ingestion of lactulose solution distinguished between COVID-19 convalescents and COVID-19 survivors with an AUC of 0.683 and 0.660, respectively. The associations of the levels of gases in the exhaled air with clinical and biochemical parameters were revealed: the presence of overweight and obesity showed inverse associations with the level of methane production (r= -0.342, p<0.05), its concentration after taking lactulose at various time intervals, and also the basic level of hydrogen (r= -0.313, p<0.05);the degree of obesity was also inversely correlated with the level of methane emission (r= -0.368, p=0.038). Direct links were established between indicators of liver elasticity in kPa and the level of hydrogen production (r=0.275, p<0.05). Conclusions. Obtained indirect signs of pronounced changes in the intestinal microbiome, which obviously contribute to a more severe course of COVID-19, the development of liver fibrosis, so the impact on the intestinal microflora can be considered as a potential target in the treatment of patients with COVID-19. © 2022 Kola Science Centre of the Russian Academy of Sciences. All rights reserved.

IN VITRO STUDY OF THE EFFECT OF CYTOFLAVIN ON ERYTHROCYTES OF PATIENTS UNDERWENT COVID-19

Erythrocytes of 40 patients (11 men and 29 women, average age 52.2 +/- 13.2 years), COVID-19 convalescents (within 2 - 4 months after the disease), were studied by dielectrophoresis using an electro-optical cell detection system in a nonuniform alternating electric field. Cytoflavin at a concentration of 1:30 (v:v) after incubation with erythrocytes (1,6 - 1,8 x 108/ml) for 10 minutes caused an increase by 24 - 63% on average in the share of discocytes, amplitude of deformation, speed of cell movement towards electrodes, magnitude of the dipole moment, capacity of the erythrocyte membranes, and in the polarizability at a frequency of 106 Hz and relative polarizability (p = 0.05 - 0.0001). On the contrary, a decrease by 27 - 49% on average is observed in the composite indicators of viscosity, cell rigidity, electrical conductivity, aggregation index, degree of hemolysis at different frequencies of the electric field (p = 0.05 - 0.002). A shift of the crossover frequency of erythrocytes (from the position of the frequency regions of negative and positive dielectrophoresis) to the low-frequency range is revealed (p < 0.0001). The analysis of data using the Volcano-plot method (paired and unpaired statistics) showed that the degree of erythrocyte deformation at a frequency of 0.5 . 106 Hz, relative polarizability, the position of the crossover frequency, membrane capacity and polarizability of cells at different frequencies of the electric field appeared to be the most sensitive to the action of cytoflavin. Thus, the action of cytoflavin triggers the processes optimizig the rheological properties of erythrocytes of patients underwent COVID-19 which is rather important for adequate micro- and macrocirculation of blood and allows expanding indications for the use of the drug in patients of this category. Copyright © 2022 Izdatel'stvo Meditsina. All rights reserved.

In Vitro Stimulation with Live SARS-CoV-2 Suggests Th17 Dominance In Virus-Specific CD4+ T Cell Response after COVID-19.

The SARS-CoV-2 and influenza viruses are the main causes of human respiratory tract infections with similar disease manifestation but distinct mechanisms of immunopathology and host response to the infection. In this study, we investigated the SARS-CoV-2-specific CD4+ T cell phenotype in comparison with H1N1 influenza-specific CD4+ T cells. We determined the levels of SARS-CoV-2- and H1N1-specific CD4+ T cell responses in subjects recovered from COVID-19 one to 15 months ago by stimulating PBMCs with live SARS-CoV-2 or H1N1 influenza viruses. We investigated phenotypes and frequencies of main CD4+ T cell subsets specific for SARS-CoV-2 using an activation induced cell marker assay and multicolor flow cytometry, and compared the magnitude of SARS-CoV-2- and H1N1-specific CD4+ T cells. SARS-CoV-2-specific CD4+ T cells were detected 1-15 months post infection and the frequency of SARS-CoV-2-specific central memory CD4+ T cells was increased with the time post-symptom onset. Next, SARS-CoV-2-specific CD4+ T cells predominantly expressed the Th17 phenotype, but the level of Th17 cells in this group was lower than in H1N1-specific CD4+ T cells. Finally, we found that the lower level of total Th17 subset within total SARS-CoV-2-specific CD4+ T cells was linked with the low level of CCR4+CXCR3- 'classical' Th17 cells if compared with H1N1-specific Th17 cells. Taken together, our data suggest the involvement of Th17 cells and their separate subsets in the pathogenesis of SARS-CoV-2- and influenza-induced pneumonia; and a better understanding of Th17 mediated antiviral immune responses may lead to the development of new therapeutic strategies.

Sulodexide in post-hospital treatment of patients with new coronavirus infection COVID-19.

Objective. To search for new effective, pathogenesis-based outpatient treatment methods for patients with new coronavirus infection (COVID-19). Material and methods. We examined 130 patients aged 45—75 years with severe COVID-19 and acute viral multisegmental pneumonia with lung tissue involvement of 30 to 63% who had previously received inpatient treatment. The patients were divided into four comparable groups according to the method of outpatient anticoagulant therapy. Group 1 (32 patients) received apixaban 2.5 mg BID for 1 month;Group 2 (32 patients) received apixaban in the same dosage for 6 months;Group 3 (32 patients) received sulodexide at 250 ULS BID and acetylsalicylic acid 100 mg/day for 1 month;Group 4 (34 patients) received sulodexide and acetylsalicylic acid in the same dosage for 6 months. Patients were examined 1, 3 and 6 months after hospital discharge. A comparative efficacy and safety analysis was performed. The criteria were the occurrence/absence of clinically significant thrombosis, changes in the coagulation tests, and the presence/absence of hemorrhagic complications in each of the treatment groups. Results. Despite the ongoing treatment, venous thrombosis occurred in 18 (13.8%) patients, mostly in the groups that stopped anticoagulant therapy after one month. Changes in coagulation tests (aPTT, INR, PTT, fibrinogen, and platelets) were statistically insignificant, except for a decrease in D-dimer, which was observed in the groups receiving anticoagulant therapy for six months. There was no statistically significant increase in hemorrhagic complications in the groups receiving continuous anticoagulant therapy. Conclusion. The data obtained suggest continuous anticoagulant therapy use (up to 6 months) in patients with severe COVID-19. (English) [ FROM AUTHOR] Цель исследования. Поиск новых эффективных, патогенетически обоснованных методов лечения пациентов с новой коронавирусной инфекцией (COVID-19) на амбулаторном этапе. Материал и методы. Обследованы 130 пациентов в возрасте 45—75 лет, предварительно получивших стационарное лечение по поводу тяжелого течения COVID-19 с развитием острой вирусной полисегментарной пневмонии с поражением легочной ткани от 30 до 63%. По методике последующей антикоагулянтной терапии на амбулаторном этапе пациенты были разделены на 4 сопоставимые группы. В 1-й группе (32 пациента) больным был назначен апиксабан по 2,5 мг 2 раза в сутки, длительностью 1 мес;во 2-й группе (32 пациента) — апиксабан в той же дозировке на протяжении 6 мес;в 3-й группе (32 пациента) — сулодексид по 250ЛЕ 2 раза в сутки и ацетилсалициловая кислота по 100 мг/сут длительностью 1 мес;в 4-й группе (34 пациента) — сулодексид и ацетилсалициловая кислота в той же дозировке в течение 6 мес. Пациенты проходили осмотр через 1, 3 и 6 мес после выписки из стационара. Был проведен сравнительный анализ эффективности и безопасности проводимого лечения, критериями которого являлись возникновение/отсутствие клинически значимых тромбозов, изменение показателей коагулограммы и наличие/отсутствие геморрагических осложнений в каждой из сравниваемых групп. Результаты. Несмотря на проводимое лечение, венозные тромбозы возникли у 18 (13,8%) пациентов — в основном в группах, прекративших антикоагулянтную терапию спустя месяц. Изменение показателей коагулограммы (АЧТВ, МНО, ПТИ, фибриноген и тромбоциты) было статистически не значимо, за исключением снижения D-димера, которое отмечено в группах, получающих антикоагулянтную терапию в течение 6 мес. В группах, получающих продолженную антикоагулянтную терапию, не произошло статистически значимого увеличения количества геморрагических осложнений. Заключение. На основании полученных данных можно говорить о целесообразности продолженной антикоагулянтной терапии (вплоть до 6 мес) у больных с тяжелым течением COVID-19. (Russian) [ FROM AUTHOR] Copyright of Profilakticheskaya Meditsina is the property of Media Sphere Publishing House and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Mild and Asymptomatic COVID-19 Convalescents Present Long-Term Endotype of Immunosuppression Associated With Neutrophil Subsets Possessing Regulatory Functions.

The SARS-CoV-2 infection [coronavirus disease 2019 (COVID-19)] is associated with severe lymphopenia and impaired immune response, including expansion of myeloid cells with regulatory functions, e.g., so-called low-density neutrophils, containing granulocytic myeloid-derived suppressor cells (LDNs/PMN-MDSCs). These cells have been described in both infections and cancer and are known for their immunosuppressive activity. In the case of COVID-19, long-term complications have been frequently observed (long-COVID). In this context, we aimed to investigate the immune response of COVID-19 convalescents after a mild or asymptomatic course of disease. We enrolled 13 convalescents who underwent a mild or asymptomatic infection with SARS-CoV-2, confirmed by a positive result of the PCR test, and 13 healthy donors without SARS-CoV-2 infection in the past. Whole blood was used for T-cell subpopulation and LDNs/PMN-MDSCs analysis. LDNs/PMN-MDSCs and normal density neutrophils (NDNs) were sorted out by FACS and used for T-cell proliferation assay with autologous T cells activated with anti-CD3 mAb. Serum samples were used for the detection of anti-SARS-CoV-2 neutralizing IgG and GM-CSF concentration. Our results showed that in convalescents, even 3 months after infection, an elevated level of LDNs/PMN-MDSCs is still maintained in the blood, which correlates negatively with the level of CD8+ and double-negative T cells. Moreover, LDNs/PMN-MDSCs and NDNs showed a tendency for affecting the production of anti-SARS-CoV-2 S1 neutralizing antibodies. Surprisingly, our data showed that in addition to LDNs/PMN-MDSCs, NDNs from convalescents also inhibit proliferation of autologous T cells. Additionally, in the convalescent sera, we detected significantly higher concentrations of GM-CSF, indicating the role of emergency granulopoiesis. We conclude that in mild or asymptomatic COVID-19 convalescents, the neutrophil dysfunction, including propagation of PD-L1-positive LDNs/PMN-MDSCs and NDNs, is responsible for long-term endotype of immunosuppression.

Detection of IFNγ-Secreting CD4+ and CD8+ Memory T Cells in COVID-19 Convalescents after Stimulation of Peripheral Blood Mononuclear Cells with Live SARS-CoV-2.

BACKGROUND: New coronavirus SARS-CoV-2, a causative agent of the COVID-19 pandemic, has been circulating among humans since November 2019. Multiple studies have assessed the qualitative and quantitative characteristics of virus-specific immunity in COVID-19 convalescents, however, some aspects of the development of memory T-cell responses after natural SARS-CoV-2 infection remain uncovered. METHODS: In most of published studies T-cell immunity to the new coronavirus is assessed using peptides corresponding to SARS-CoV-1 or SARS-CoV-2 T-cell epitopes, or with peptide pools covering various parts of the viral proteins. Here, we determined the level of CD4+ and CD8+ memory T-cell responses in COVID-19 convalescents by stimulating PBMCs collected 1 to 6 months after recovery with sucrose gradient-purified live SARS-CoV-2. IFNγ production by the central and effector memory helper and cytotoxic T cells was assessed by intracellular cytokine staining assay and flow cytometry. RESULTS: Stimulation of PBMCs with live SARS-CoV-2 revealed IFNγ-producing T-helper effector memory cells with CD4+CD45RA-CCR7- phenotype, which persisted in circulation for up to 6 month after COVID-19. In contrast, SARS-CoV-2-specific IFNγ-secreting cytotoxic effector memory T cells were found at significant levels only shortly after the disease, but rapidly decreased over time. CONCLUSION: The stimulation of immune cells with live SARS-CoV-2 revealed a rapid decline in the pool of effector memory CD8+, but not CD4+, T cells after recovery from COVID-19. These data provide additional information on the development and persistence of cellular immune responses after natural infection, and can inform further development of T cell-based SARS-CoV-2 vaccines.