COVID-19. Aetiology, pathogenesis, diagnosis and treatment

COVID-19 (Coronavirus disease 2019) is a new epidemic infectious disease characterized by a relatively high contagiousness and a high probability of life-threatening complications such as acute respiratory distress syndrome (ARDS), acute respiratory and multiple organ failure. The causative agent of the disease is the enveloped zoonotic RNA virus known as SARS-CoV-2. Together with the SARS-CoV and MERS-CoV viruses, which cause severe acute respiratory syndrome and the Middle East respiratory syndrome, respectively, it belongs to the Coronaviridae family, the genus Betacoronavirus. The COVID-19 epidemic has spread rapidly around the world and currently hit 213 countries with more than 1.6 million confirmed cases, of which more than 90.000 have died so far. In Russian Federation, SARS-CoV-2 infection is included in the list of diseases that pose a danger to others, along with especially dangerous infections. The virus is transmitted by airborne droplets, airborne dust and contact routes, therefore, to protect medical staff it is necessary to use individual protective suits and accessories, with protection of the respiratory tract and eyes, disinfection of hands and surfaces as when working with the BSL-2 microorganisms. The diagnosis of COVID-19 is confirmed using real-time RT-PCR diagnostics detecting the presence of viral RNA. Clinical manifestations of COVID-19 vary from mild and moderate (pneumonia without signs of hypoxemia and impaired O2 saturation, 80% of all cases), to severe (15% of cases, O2 saturation 8993%) and extremely severe (5% of cases, ARDS, multiple organ failure, mechanical ventilation and resuscitation are necessary). The typical clinical presentation of COVID19 patients includes the following: severe fever, dry cough, respiratory failure, combined with lymphopenia and thrombocytopenia, normal procalcitonin, elevated levels of ferritin and CRP in the blood with signs of bilateral, polysegmental pneumonia and the ground glass opacity on CT. Even in the absence of an unfavorable epidemiological anamnesis, these clinical signs can be recommended to admit the patient to an infectious isolation ward where he or she would wait for the results of PCR diagnostics and the diagnosis of COVID-19 be confirmed/ ruled out. Currently, no SARS-CoV-2-specific therapy is available for COVID-19 patients; the only method that has proven effective in several investigational trials is transfusion of convalescent plasma with high titers of neutralizing antibodies. A number of innovative treatments appear promising and include the use of neutralizing monoclonal antibodies, ACE2-derived agents, as well as MSC- and NK-cell based cell therapies

Surgical treatment of patients with symptomatic Kimmerle's anomaly using video endoscopy

Background: Clinical manifestations of Kimmerle’s anomaly are detected in 5.5 to 20% of patients. The main reason for the development of symptoms is prolonged compression of the V3 (atlantic) segment of the vertebral artery in the bone ring as a result of the atlantooccipital membrane’s exostosis. To date, the final tactics for treating patients with Kimmerle’s anomaly has not been determined. The effectiveness of conservative methods of therapy does not exceed 40%. The aim Of this study was to evaluate the results of a minimally invasive surgical treatment of patients with symptomatic Kimmerle’s anomaly using video endoscopic assistance. Methods: In the period from 2020 to 2022, 15 patients were operated on. The indication for the surgical treatment was the lack of the conservative therapy’s effect for 1 year from the onset of the disease, aggravation of the disease symptoms, a decrease in the blood flow through the vertebral artery at the Kimmerle’s anomaly side when turning the head. The vertebral artery decompression was performed using video endoscopy through a posterior median approach in two (13%) patients and through a paravertebral intermuscular approach (4 cm incision in the occipitocervical region in the projection of the Kimmerle’s anomaly) in thirteen (87%) patients. Results: The outcome of the disease was assessed at the time of discharge from the hospital, as well as in 6 and in 12 months after the operation. Following the surgical treatment, all the patients showed the complete regression of symptoms and restoration of the blood flow velocities in the vertebral artery. There were no complications after the operation. The use of video endoscopy made it possible to reduce the size of the surgical wound from 12 cm to 4 cm, which contributed to a decrease in the intensity of pain in the postoperative period, early activation and a decrease in the duration of the inpatient treatment. Conclusion: With the proper selection of patients with Kimmerle’s anomaly, decompression of the V3 segment of the VA using video endoscopy is a safe and effective method of treatment

Umbilical cord blood as a promising source of NK cells for immunotherapy

Currently, a large number of studies on genetic modification of cord blood NK cells (UCB-NK) are carried out at both clinical and preclinical levels. Immunotherapy based on UCB-NK cells has great potential for antitumor therapy. However, despite having known several advantages over peripheral blood NK cells (PB- NK), including a high concentration in cord blood and low virulence rate, UCB-NK cells are predominantly characterized in the scientific literature as immature and low-functioning NK cells. In this work, we studied the phenotypic characteristics of UCB-NK cells and the possibility of stimulatory compensation of the decreased functional activity of UCB-NK cells. Our studies revealed UCB-NK cells can be characterized as poorly differentiated and weakly activated cells with high level of inhibitory receptor NKG2A and low level of activating receptor NKG2C and HLA-DR, accordingly with the literature data. Two types of stimuli were chosen to stimulate freshly isolated UCB-NK cells: 1) 100 units of IL-2; 2) combinations of 100 units IL-2 and K-562 feeder cells expressing membrane-bound IL-21 (K562-mbIL21). It was shown the degranulation (LAMP-1) and proliferative activity was higher than for parallel cultured ex vivo PB-NK cells under the same conditions for UCB-NK cells stimulated for 7 days with IL-2 + K562-mbIL21. Moreover, stimulation in the way of IL-2 + K562-mbIL21 seemed to be a more perspective way to obtain a large number of proliferatively active UCB-NK cells compared to stimulation with IL-2 only. Since genetic modification of NK cells is a promising way to improve the antitumor properties of NK cells, retroviral transduction procedure was performed to study of the stimulated UCB-NK cells. UCB-NK cells stimulated with IL-2 + K562-mbIL21 were transduced on day 8 of cultivation. In this study, we used targeted overexpression of the adaptor molecule DAP12, which is involved in the signaling of activating NK cell receptors. PB-NK cells and UCB-NK cells were transduced under the equal experimental conditions in same volume of viral particles. As a result, the transduction efficiency was found to be more than 4-fold higher for UCB-NK cells compared to PB-NK cells. Thus, UCB-NK cells appear to be a promising tool for further research in cancer immunotherapy

Long-term results of microvascular decompression with video endoscopy in the treatment of patients with atypical trigeminal neuralgia

Background: The incidence of atypical trigeminal neuralgia (aNTN) varies from 1 to 7 per 100,000 population per year. The main cause of its development is compression of the trigeminal nerve (TN) root by a vein and/or artery in the cerebellar cistern. To date, the final tactics of treatment for patients with aNTN has not been specified. The effectiveness of conservative methods of therapy does not exceed 50%. The aim of this study was to evaluate the results of microvascular decompression using video endoscopy in the treatment of patients with atypical trigeminal neuralgia. Methods: In the period from 2014 to 2021, 34 patients with aNTN were operated on, of which 18 (53%) patients had neuropathic pain (more than 4 points on the DN4 scale), and 15 (44%) patients had transformation of classical trigeminal neuralgia into atypical neuralgia. The conservative therapy (carbamazepine, gabapentin, pregabalin), administered to all the patients in the preoperative period, was not accompanied by a significant relief of pain syndrome. The maximum intensity of pain upon admission to the hospital was, according to the visual analog scale (VAS), 10 points, according to the BNI (Barrow Neurological Institute) Pain Intensity Scale V (severe, persistent pain). All the patients underwent microvascular decompression of the trigeminal nerve root with the use of Teflon; in 12 (35%) patients, in addition to microscopy, video endoscopy was used. The average follow-up period after the surgery was 3.41.7 years (from 1 to 5 years). Results: In all (100%) patients, the pain was completely eliminated (BNI I) after the surgery. A total five-year excellent and good outcome of the disease on the J. Miller and BNI scale (I -II) was noted in 80% (n=27) of patients with aNTN. The risk of pain recurrence after microvascular decompression was 14% (n=3) in the first three years, and 34% (n=4) after 5 years. The use of video endoscopy made it possible to identify the blood vessels compressing the root of the trigeminal nerve with a minimal displacement of the cerebellum and cranial nerves when visualizing the neurovascular conflict. Conclusion: The microvascular decompression method with video endoscopy is effective in the treatment of patients with aNTN

Tumor inflating lymphocytes. Purification, expanding and cytotoxicity analisys on primary tumor cultures

Background. Tumor Infiltrating Lymphocytes (TILs) is one of the most promising sources of autologous cytotoxic T-cells for adoptive immunotherapy, which has already shown high efficiency in the treatment of metastatic melanoma. However, the isolation of TILs from solid tumors is technically difficult. A suppressive tumor microenvironment, in particular, a high level of expression of check-point inhibitors PD-1 CTLA4, tissue hypoxia and other factors cause that T cells isolated from the tumor do not proliferate well and do not exhibit cytotoxic properties. Aims. In this study, we isolated TILs from surgical material obtained by resection of solid tumors (primary and metastatic adenocarcinomas of various localization, melanoma, glioblastoma), studied their population composition and developed protocols for the purification expanding, and activation of CD4+, CD8+ cytotoxic antitumor lymphocytes. Methods. An urgent task is the activation of TILs, turning off immunosuppressive mechanisms and increasing their antitumor cytotoxic activity. Various approaches are used for this: activation by a cocktail of cytokines and antibodies, editing the lymphocyte genome by knocking out suppressor genes or, conversely, transduction of activating genes, coincubation with feeder cells, etc. Cells were obtained from samples of resected tumors in 16 patients; in each case we obtain an autologous pair: the primary tumor culture and the TILs culture. Results. We could isolate viable lymphocytes in 100% of cases. Isolated TILs were successfully expanded in our specialized medium using various combinations of IL-2, IL-15, IL-21, IL-7, anti-CD3 and anti-CD28. Immunophenotyping showed that the obtained TILs are a heterogeneous mixture of CD4+, CD8+ cells containing populations of CD3+CD8+CD45+(CTL) CD3+CD4+CD45+ (T-helpers), CD4+CD25+CD127- (Т-regulatory cells), CD3-CD56+CD45+ (NK-cells), CD3+CD56+CD45+ (Т-NK-cells). The initial cultures of TILs were also characterized by a high level of PD1 expression, indicating their low antitumor cytotoxicity. Using different protocols of isolation, expansion, and activation, we obtained a cell preparation containing 80% of CD8+ PD-1- activated TILs in an amount sufficient for adoptive therapy (500106 or more). An in vitro study of the cytotoxicity of obtained TILs in primary cultures of homologous tumors using RTCA Icelligence showed high cytotoxicity, providing almost 100% tumor cell death. Conclusion. Our developed protocol for the production and activation of TILs can be recommended for the phase III clinical trials of adoptive immunotherapy of recurrent, highly metastatic solid tumors

РАЗРАБОТКА И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ НЕЙРОПОРТА ДЛЯ ТЕРАПИИ ГЛИОМЫ ГОЛОВНОГО МОЗГА

Current paper presents the results of the system development for intracranial implantation aimed on therapy and prevention of brain gliomas relapse. The main property of the system, in prospective,  will be to direct the growth of glioma cells localized in the region  adjacent to the site of the removed tumor along the fi bers towards  the proximal part of the fiber-optic scaffold (neuroport). Such  approach will allow carrying out cells diagnostics by the  photoluminescence signal and provide subsequent destruction of  malignant cells by photodynamic action. Besides, this system could  be used for monitoring the processes occurring in the probed area in order to control the possible relapses. The localization of cells along  the fi ber structures covered with gelatin compound, which is the  source of amino acids during cultivation, was shown during the glioma cells growth dynamics study. Moreover, four different designs of intracranial scaffold models, serving as ports for diagnostic and therapeutic laser radiation delivery, were developed and  successfully tested in the framework of the research. The results  obtained on the rats brain with induced tumors (glioma C6) after  neuroport implantation demonstrate sufficiently intense fluorescence in the tumor bed after intravenous injection of the  nonmetallic sulfonated phthalocyanine based photosensitizer, and a  pronounced photodynamic effect leading to total destruction of the  tumor. In this way, the results of this study open the prospects of creating the neuroport with an internal fi ber structure that focuses the glioma cells growth.В работе представлены результаты разработки системы для внутричерепной имплантации с целью терапии и предотвращения рецидивирования глиом головного. Основное свойство  системы в перспективе будет состоять в том, чтобы направить рост клеток глиомы,  локализованных в области, прилегающей к месту удаленной опухоли, вдоль волокон по  направлению к проксимальной части волоконно-оптического имплантата (нейропорт) с  целью их регистрации по сигналу фотолюминесценции и последующей их деструкции в  результате фотодинамического воздействия. Такое устройство должно обеспечить  мониторинг процессов, происходящих в зондируемой области с целью контроля процессов  рецидивирования. В ходе данного исследования динамики роста клеток глиомы показана  локализация клеток вдоль волоконных структур, покрытых желатином, который является  источником аминокислот при культивировании. Также в ходе работы были разработаны и  успешно апробированы четыре различных конструкции макетов внутричерепных  имплантатов, выполняющие роль портов для доставки диагностического и терапевтического лазерного излучения. Получены на головном мозге крыс с индуцированными опухолями  (глиома С6) после имплантации нейропорта, демонстрирующие достаточно интенсивную  флуоресценцию в ложе опухоли при внутривенном введении фотосенсибилизатора на  основе безметального сульфированного фталоцианина и выраженный фотодинамический  эффект, приведший к тотальному разрушению опухоли. Полученные результаты открывают  перспективы создания нейропорта с внутренней волоконной структурой, фокусирующей рост клеток глиомы

Safety and efficacy of convalescent plasma for COVID-19: the preliminary results of a clinical trial

Background. The lack of effective etiotropic therapy for COVID-19 has prompted researchers around the globe to seekr various methods of SARS-CoV-2 elimination, including the use of convalescent plasma. Aim. The aim of this work was to study the safety and efficacy of the convalescence plasma treatment of severe COVID-19 using the plasma containing specific antibodies to the receptor binding domain (RBD) of SARS-CoV-2 S protein in a titer of at least 1:1000. Methods. A single-center, randomized, prospective clinical study was performed at the FRCC FMBA of Russia with the participation of 86 patients who were stratified in two groups. The first group included 20 critically ill patients who were on mechanical ventilation the second group included 66 patients with moderate to severe COVID-19 and with spontaneous respiration. The patients in the second group were randomized into two cohorts in a ratio of 2:1. In the first cohort (46 patients), pathogen-reduced convalescent plasma was transfused (twice, 320 ml each), in the second cohort (20 patients) a similar amount of non-immune freshly frozen plasma was transfused to the patients. Results. The use of plasma of convalescents in patients with severe COVID-19 being on mechanical ventilation does not affect the disease outcome in these patients. The mortality rate in this group was 60%, which corresponds to the average mortality of COVID patients on mechanical ventilation in our hospital. In the second group, clinical improvement was detected in 75% and 51%, for convalescent and non-immune plasma, respectively. Of the 46 people who received convalescent plasma, three patients (6.5%) were transferred to mechanical ventilation, two of them died. In the group receiving non-immune plasma, the need for mechanical ventilation also arose in three patients (15%), of which two died. The hospital mortality in the group of convalescent plasma was 4.3%, which is significantly lower than the average COVID-19 hospital mortality at our Center (6.73%) and more than two times lower than the hospital mortality in the control group (n=150), matched by age and by the disease severity. Conclusions. Thus, we demonstrated a relative safety of convalescent plasma transfusion and the effectiveness of such therapy for COVID-19 at least in terms of the survival of hospitalized patients with severe respiratory failure without mechanical ventilation. In the absence of bioengineered neutralizing antibodies and effective etiotropic therapy, the use of hyperimmune convalescent plasma is the simplest and most effective method of specific etiopathogenetic therapy of severe forms of COVID-19

Association between convalescent plasma treatment and mortality in COVID-19: a collaborative systematic review and meta-analysis of randomized clinical trials.

Funder: laura and john arnold foundationBACKGROUND: Convalescent plasma has been widely used to treat COVID-19 and is under investigation in numerous randomized clinical trials, but results are publicly available only for a small number of trials. The objective of this study was to assess the benefits of convalescent plasma treatment compared to placebo or no treatment and all-cause mortality in patients with COVID-19, using data from all available randomized clinical trials, including unpublished and ongoing trials (Open Science Framework, https://doi.org/10.17605/OSF.IO/GEHFX ). METHODS: In this collaborative systematic review and meta-analysis, clinical trial registries (ClinicalTrials.gov, WHO International Clinical Trials Registry Platform), the Cochrane COVID-19 register, the LOVE database, and PubMed were searched until April 8, 2021. Investigators of trials registered by March 1, 2021, without published results were contacted via email. Eligible were ongoing, discontinued and completed randomized clinical trials that compared convalescent plasma with placebo or no treatment in COVID-19 patients, regardless of setting or treatment schedule. Aggregated mortality data were extracted from publications or provided by investigators of unpublished trials and combined using the Hartung-Knapp-Sidik-Jonkman random effects model. We investigated the contribution of unpublished trials to the overall evidence. RESULTS: A total of 16,477 patients were included in 33 trials (20 unpublished with 3190 patients, 13 published with 13,287 patients). 32 trials enrolled only hospitalized patients (including 3 with only intensive care unit patients). Risk of bias was low for 29/33 trials. Of 8495 patients who received convalescent plasma, 1997 died (23%), and of 7982 control patients, 1952 died (24%). The combined risk ratio for all-cause mortality was 0.97 (95% confidence interval: 0.92; 1.02) with between-study heterogeneity not beyond chance (I2 = 0%). The RECOVERY trial had 69.8% and the unpublished evidence 25.3% of the weight in the meta-analysis. CONCLUSIONS: Convalescent plasma treatment of patients with COVID-19 did not reduce all-cause mortality. These results provide strong evidence that convalescent plasma treatment for patients with COVID-19 should not be used outside of randomized trials. Evidence synthesis from collaborations among trial investigators can inform both evidence generation and evidence application in patient care

Functional Profiling of In Vitro Reactivated Memory B Cells Following Natural SARS-CoV-2 Infection and Gam-COVID-Vac Vaccination

Both SARS-CoV-2 infection and vaccination have previously been demonstrated to elicit robust, yet somewhat limited immunity against the evolving variants of SARS-CoV-2. Nevertheless, reports performing side-by-side comparison of immune responses following infection vs. vaccination have been relatively scarce. The aim of this study was to compare B-cell response to adenovirus-vectored vaccination in SARS-CoV-2-naive individuals with that observed in the COVID-19 convalescent patients six months after the first encounter with the viral antigens. We set out to use a single analytical platform and performed comprehensive analysis of serum levels of receptor binding domain (RBD)-specific and virus-neutralizing antibodies, frequencies of RBD-binding circulating memory B cells (MBCs), MBC-derived antibody-secreting cells, as well as RBD-specific and virus-neutralizing activity of MBC-derived antibodies after Gam-COVID-Vac (Sputnik V) vaccination and/or natural SARS-CoV-2 infection. Overall, natural immunity was superior to Gam-COVID-Vac vaccination. The levels of neutralizing MBC-derived antibodies in the convalescent patients turned out to be significantly higher than those found following vaccination. Our results suggest that after six months, SARS-CoV-2-specific MBC immunity is more robust in COVID-19 convalescent patients than in Gam-COVID-Vac recipients. Collectively, our data unambiguously indicate that natural immunity outperforms Gam-COVID-Vac-induced immunity six months following recovery/vaccination, which should inform healthcare and vaccination decisions

Functional Profiling of In Vitro Reactivated Memory B Cells Following Natural SARS-CoV-2 Infection and Gam-COVID-Vac Vaccination

Both SARS-CoV-2 infection and vaccination have previously been demonstrated to elicit robust, yet somewhat limited immunity against the evolving variants of SARS-CoV-2. Nevertheless, reports performing side-by-side comparison of immune responses following infection vs. vaccination have been relatively scarce. The aim of this study was to compare B-cell response to adenovirus-vectored vaccination in SARS-CoV-2-naive individuals with that observed in the COVID-19 convalescent patients six months after the first encounter with the viral antigens. We set out to use a single analytical platform and performed comprehensive analysis of serum levels of receptor binding domain (RBD)-specific and virus-neutralizing antibodies, frequencies of RBD-binding circulating memory B cells (MBCs), MBC-derived antibody-secreting cells, as well as RBD-specific and virus-neutralizing activity of MBC-derived antibodies after Gam-COVID-Vac (Sputnik V) vaccination and/or natural SARS-CoV-2 infection. Overall, natural immunity was superior to Gam-COVID-Vac vaccination. The levels of neutralizing MBC-derived antibodies in the convalescent patients turned out to be significantly higher than those found following vaccination. Our results suggest that after six months, SARS-CoV-2-specific MBC immunity is more robust in COVID-19 convalescent patients than in Gam-COVID-Vac recipients. Collectively, our data unambiguously indicate that natural immunity outperforms Gam-COVID-Vac-induced immunity six months following recovery/vaccination, which should inform healthcare and vaccination decisions