Mucosal epithelial cells and novel approaches to immunoprophylaxy and immunotherapy of infectious diseases
Innate immune defense mechanisms activated by pathogen recognition can be roughly divided into two categories: 1) those aimed at direct killing of the pathogen (microbicidal); 2) those aimed at the development of inflammation (pro-inflammatory). The final goal of both types of mechanisms is elimination of the pathogen and restoration of homeostasis. However, inflammation is accompanied by damage of self tissues, which often has a negative impact on the outcome of infection. Therefore, there is a need in such approaches to immunoprophylaxy and immunotherapy of infections that would enhance antimicrobial defense while minimizing inflammation. A key approach is elevation of epithelial barrier resistance. The proposed concept of immunostimulation is exemplified using COVID-19 infection.
IL-21/CD40L stimulation of human B-lymphocytes in vitro and their characteristics
Introduction. Cell lines and cultures are indispensable tools in cell physiology research. Cultures of B lymphocytes are essential for the study of B-cell response, antibody generation, as well as for the creation of therapeutic monoclonal antibodies. At the same time, the activation of B-lymphocytes and their long-term cultivation in vitro continues to be an unsolved problem.
The aim of the study - development of a protocol for the stimulation of human B lymphocytes in vitro, characterization of the phenotype and functional characteristics of stimulated B cells for the subsequent use of the protocol for determining the number of memory B cells, sequencing of Ig genes, as well as obtaining immortalized clones of B lymphocytes.
Material and methods. Cells stably transfected with the CD40L gene were generated by lentiviral transduction. B lymphocytes were isolated from human peripheral blood by centrifugation in a density gradient of ficoll-verografin with further enrichment by negative selection using magnetic beads. B lymphocytes were stimulated in vitro with feeder cells carrying surface CD40L in the presence of exogenous recombinant interleukin(IL)-21. The number and phenotype of stimulated B lymphocytes were determined using multicolor flow cytometry. The secretion of IgM and IgG in cultures of B lymphocytes was assessed using enzyme-linked immunosorbent assay.
Results. Using lentiviral transduction, HEK293, K562 and A549 cells stably transfected with the CD40L gene were obtained. Based on the expression of the CD40L molecule, as well as growth properties, the transfected A549 cell line was selected for further research as a feeder. When feeder cells and B lymphocytes were co-cultured in the presence of exogenous IL-21, stimulation of B lymphocytes was observed. B lymphocytes began to proliferate and after 7 days their number increased by an average of 8 times. The stimulated B lymphocytes changed their morphology. They acquired an irregular shape with pseudopodia, grouped around feeder cells, and began to move actively. Upon IL-21/CD40L stimulation, B lymphocytes changed their surface phenotype and by day 10, about 40 % of B cells had differentiated into plasmablasts (CD19+CD27+CD38+). At the same time, the proportion of CD20+ cells decreased to 20 %. The percentage of cells with surface Ig expression also decreased. In contrast, the secretion of IgM and IgG increased during stimulation. By the 12th day, the proliferative potential of B cells was exhausted, and they died.
Сonlusion. In vitro cultivation of B lymphocytes in the presence of exogenous IL-21, as well as feeder cells expressing the CD40L molecule, is a convenient system for obtaining activated B lymphocytes that can be used in different applications.
The HLA-Cw6 allele as the genetic predisposing factor to psoriasis in Tatar population
Introduction. Psoriasis is an immune-mediated and multifactorial disease with a strong genetic background. HLA-Cw6 is one of the most strongly associated psoriasis susceptibility alleles.
Aim of the study was to assess the association allele HLA-Cw6 of HLA-C gene with psoriasis in Tatar patients with psoriasis in Volga-Ural region.
Material and methods. A case-control genetic association study was carried out. DNA samples obtained from a total of 106 cases and 175 healthy donors were examined. Genotyping allele HLA-Cw6 of the HLA-C gene was performed by PCR-RFLP (polymerase chain reaction-polymorphism of the lengths of restriction fragments).
Results. The presence of HLA-Cw6 allele was associated with psoriasis, being more frequent for patients with disease vs group of comparison (47 % и 16 %, respectively) (OR = 4.5; 95 % CI 2.85-7.15; p < 0.0005).
Conclusion. The present findings suggest that allele HLA-Cw6 of HLA-C gene contributes to the genetic susceptibility to psoriasis in the Tatar population.
Protective capabilities of IL-2 in recovery after traumatic brain injury in animals of different ages
Introduction. Elder age is a significant negative health factor after traumatic brain injury (TBI).
Aim was to study the dynamics of the brain-derived neurotrophic factor (BDNF) level, immune and hormonal disturbances that develop in young and elderly rats during the recovery period after TBI with the correction by recombinant interleukin (IL)-2 (rIL-2).
Material and methods. The work was performed on male Wistar rats aged 3 (young) and 18 months (elderly). The «weight drop» model was used as a model for TBI. To correct disturbances caused by TBI animals were medicated with commercial recombinant rIL-2 («Biotech», Russian Federation) in dose of 30 mcg/kg of body weight after the TBI once a day for 3 days. Control animals were injected with 0.15 М NaCl. On days 7 and 14 after TBI, the concentrations of corticosterone, testosterone and BDNF in the blood of animals were determined by ELISA; the activity of immune cells was determined by their cytotoxicity and proliferative activity.
Results. In intact and injured animals, both young and elderly, increased concentrations of corticosterone and testosterone were observed after administration of rIL-2 compared to animals not receiving rIL-2. Administration of rIL-2 to intact young and elderly animals did not change the BDNF concentration. TBI led to a significant decrease in BDNF levels in young and old animals, but BDNF levels significantly increased after rIL-2 administration in TBI groups (in both groups p < 0.05). After TBI the proliferative activity of splenocytes in young rats did not decrease, while in elderly rats the inhibition of the proliferative activity of splenocytes was significant (the stimulation index was below one). Administration of rIL-2 did not change the proliferative activity of splenocytes in young intact and injured rats. In elderly animals, a significant increase of suppressed proliferative splenocytes activity after TBI was observed; but in intact animals, the proliferative activity of lymphocytes did not change. TBI led to inhibition of the cytotoxic activity of splenocytes in young and elderly animals; the administration of rIL-2 increased it in injured rats, but did not change it in intact young and elderly rats.
Conclusion. rIL-2 in significand degree reversed the posttraumatic changes. Thus, exogenic rIL-2 may to some degree mitigate elder-age-related disfunctions caused and boosted by TBI.
Prophylaxis of medium and severe clinical forms of COVID-19 infection with aminodihydrophtalazindione sodium salt (Galavit®) in medical personnel working in the «red zone»
Introduction. Nowadays the aspects of COVID-19 prophylaxis remain especially actual among medical personnel providing medical aid to patients with this disease, mainly because of high risk of contact with the pathogen. Aminodihydrophtalazindione sodium salt is one of promising drugs for providing such prophylaxis.
Aim. Studying the effectiveness of medium and severe clinical forms of COVID-19 prophylaxis with the drug mentioned among medical personnel working in the «red zone» under circumstances of high epidemiological risk of COVID-19.
Material and methods. The study is retrospective and observational. 250 participants are included in the study. Statistical analysis was conducted using Pearson’s χ2 test, Mann-Whitney (U-test, multiple linear regression for creating the mathematical model of medium and severe COVID-19 forms risk determination.
Results. Statistically significant lowering of the risk of medium and severe COVID-19 forms is shown for prophylaxis with sodium aminodihydrophtalazindione; no effect on PCR test result was shown, as well as on quantitative grade of immune response (IgG concentration). In mathematical model predicting medium and severe forms statistical significance was found for the fact of prophylaxis and the positive immunological test (with IgG concentration higher than the threshold), that confirms the fact of contact with SARS-CoV-2.
Conclusion. A statistically significant reduction of the risk of moderate and severe forms of COVID-19 development was shown during prophylaxis with the investigated drug.
Correction of mucosal immunity and antiviral therapy of frequently recurrent chronic tonsillitis/pharyngitis with local replication of herpes viruses
Introduction. Insufficient effectiveness of traditional methods of treatment of chronic tonsillitis/pharyngitis (CT/CF), leads to the need for a deeper study of the ethio- and immuno-pathogenesis of these conditions and the search for new approaches to the treatment of such patients.
Aim - to study the features of mucosal immunity in patients with chronic often recurrent oropharyngeal inflammation in combination with local replication of herpes group viruses (Ep-stein-Barr virus, Human Herpes Virus 6, Cytomegalovirus) and to evaluate the clinical and laboratory effectiveness of combined therapy.
Material and methods. The study included 70 patients who received combination therapy. Before and after treatment, they conducted questionnaires, oropharyngeal culture for bacterial flora, quantitative determination of herpes virus DNA, and determination of the concentration of α-defensins (HNP1-3), catelicidin LL-37, lactoferrin, and sIgA in saliva.
Results. On the background of treatment was a reduction in the proportion of pathogenic microflora, the rate of detection and viral load herpes viruses, recovery factors, mucosal immunity in the oropharynx of patients of the study group, as well as decrease the frequency of relapses HT/HF in a year after treatment.
Conclusion. The clinical and laboratory effectiveness of combined therapy of frequently recurrent CT/CF has been proved.
Factors associated with variability of the course of X-linked agammaglobulinemia
Introduction. Historically the development of a section of clinical immunology dedicated to the problem of primary immunodeficiencies (PID) has begun with X-linked agammaglobulinemia (XLA). Despite numerous studies and achievements in the diagnosis and treatment of XLA, not all the problems associated with the management of patients with this variant of PID have been resolved. Evidence of Btk effect on the functions of not only B lymphocytes, but also other components of the immune system determines interest for the complex characterization of cellular factors of the innate and adaptive immune response.
The aim of the study was to determine the changes in the cellular parameters of the immune response and their dynamics depending on the duration and severity of the course of X-linked agammaglobulinemia.
Material and methods. 12 patients with XLA were under dynamic observation. ВТК genetic defect confirmed using next generation sequencing technology. In immunological monitoring, methods were used to assess the quantitative and functional parameters of the factors of innate and adaptive immunity.
Results. The results indicate that in patients with XLA, the processes of maturation and cytotoxicity of T-effectors are enhanced with inhibition of immunoregulatory suppression, neutrophil microbicidal activity, and natural killer cytotoxicity. It was shown that a more severe course is associated with a more significant inhibition of neutrophil metabolism; an increase in the experience of the disease is associated with an increase in the number of T-effectors, a decrease in the cytolytic potentials of natural killers.
Conclusion. The obtained results undoubtedly require further study in a larger cohort of patients. Nevertheless, data on the quality and degree of involvement of the cellular components of the immune response should be considered when managing patients with XLA in daily practice.
Autorestriction and resolution of allergic process
Allergen-specific inflammation is a particular manifestation of inflammation, a universal form of reactivity aimed at eliminating damage and restoring homeostasis. Modern data are presented that confirm the idea that the natural resolution of inflammation is an active process performed by coordinated cellular reactions. This process is induced and carried out by the action of anti-inflammatory mediators of various chemical nature and specialized lipid mediators. The mechanism for restriction and resolving inflammation includes inactivation of pro-inflammatory mediators, limitation of further recruitment and activation of inflammatory cells, apoptosis of these cells, switching of macrophages from M1 to M2 type, enhancement of efferocytosis, promoting the return of cells that have not undergone apoptosis to lymphatic and blood vessels, and initiation of tissue repair. Insufficiency or loss of anti-inflammatory and pro-resolving functions leads to prolongation of the inflammatory reaction, chronic inflammation, tissue remodeling and allergen-nonspecific tissue hyperreactivity. Restoration of insufficient or lost resolving function is a strategically justified task of creating new therapeutic approaches.