Analysis of centrosomal area actin reorganization and centrosome polarization upon lymphocyte activation at the immunological synapse

T cell receptor (TCR) and B cell receptor (BCR) stimulation of T and B lymphocytes, by antigen presented on an antigen-presenting cell (APC) induces the formation of the immunological synapse (IS). IS formation is associated with an initial increase in cortical filamentous actin (F-actin) at the IS, followed by a decrease in F-actin density at the central region of the IS, which contains the secretory domain. This is followed by the convergence of secretion vesicles towards the centrosome, and the polarization of the centrosome to the IS. These reversible, cortical actin cytoskeleton reorganization processes occur during lytic granule secretion in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, proteolytic granules secretion in B lymphocytes and during cytokine-containing vesicle secretion in T-helper (Th) lymphocytes. In addition, several findings obtained in T and B lymphocytes forming IS show that actin cytoskeleton reorganization also occurs at the centrosomal area. F-actin reduction at the centrosomal area appears to be associated with centrosome polarization. In this chapter we deal with the analysis of centrosomal area F-actin reorganization, as well as the centrosome polarization analysis towards the IS.

Prognosis of severe cytomegalovirus infection in newborns

Objective is to study the features of impaired activation of T and B lymphocytes in order to predicting severe cytomegalovirus infection in newborns. Materials and methods. 133 newborns with cytomegalovirus infection were examined. Immediately after diagnosing cytomegalovirus infection, all patients observed were immunologically ex amined, including assessing count of peripheral blood T and B lymphocytes, as well as their intercellular interaction by using flow cytometry immunostaining for CD3, CD3+CD28–, CD3+CD28+, CD3–CD28+, CD4, CD8, CD20, CD20+CD40+, CD28, CD40. The test was performed by using a Beckman Coulter Epics XL laser flow cytofluorometer. Depending on the condition severity, all children were divided into two groups: 1 — cytomegalovirus infection, severe form — 60 subjects (45.1%); 2 — cytomegalovirus infection, moderate form — 73 subjects (54.9%). Results of the entire set of studied indicators for cellular and humoral arms of immune system revealed statistically significant differences for the prognosis of severe cytomegalovirus infection: CD3+CD28–, CD20, CD20+CD40+, CD4. T lymphocytes with CD3+CD28+ activation markers, through which costimulating signals necessary for the activation of T helper cells are exerted cell-intrinsic features, serving as an important factor ensuring immune response. Using the “classification trees” method, we developed a differentiated approach to forecast severe cytomegalovirus infection in newborns. Systems of inequalities were obtained, four of which classify a subgroup of newborns with severe cytomegalovirus infection. The consistent application of the obtained inequalities makes it possible to isolate from the input stream of sick patients with a prognosis of the development of severe cytomegalovirus infection. The proposed diagnostic rules can be considered as screening markers for predicting a severe cytomegalovirus infection in newborns, which makes possible the timely onset of specific therapy.

A non-bactericidal cathelicidin provides prophylactic efficacy against bacterial infection by driving phagocyte influx

The roles of bactericidal cathelicidins against bacterial infection have been extensively studied. However, the anti-bacterial property and mechanism of action of non-bactericidal cathelicidins are rarely known. Herein, a novel naturally occurring cathelicidin (PopuCATH) from tree frog (Polypedates puerensis) didn’t show any direct anti-bacterial activity in vitro. Intriguingly, intraperitoneal injection of PopuCATH before bacterial inoculation significantly reduced the bacterial load in tree frogs and mice, and reduced the inflammatory response induced by bacterial inoculation in mice. PopuCATH pretreatment also increased the survival rates of septic mice induced by a lethal dose of bacterial inoculation or cecal ligation and puncture (CLP). Intraperitoneal injection of PopuCATH significantly drove the leukocyte influx in both frogs and mice. In mice, PopuCATH rapidly drove neutrophil, monocyte/macrophage influx in mouse abdominal cavity and peripheral blood with a negligible impact on T and B lymphocytes, and neutrophils, monocytes/macrophages, but not T and B lymphocytes, were required for the preventive efficacy of PopuCATH. PopuCATH did not directly act as chemoattractant for phagocytes, but PopuCATH obviously drove phagocyte migration when it was cultured with macrophages. PopuCATH significantly elicited chemokine/cytokine production in macrophages through activating p38/ERK mitogen-activated protein kinases (MAPKs) and NF-κB p65. PopuCATH markedly enhanced neutrophil phagocytosis via promoting the release of neutrophil extracellular traps (NETs). Additionally, PopuCATH showed low side effects both in vitro and in vivo. Collectively, PopuCATH acts as a host-based immune defense regulator that provides prophylactic efficacy against bacterial infection without direct antimicrobial effects. Our findings reveal a non-bactericidal cathelicidin which possesses unique anti-bacterial action, and highlight the potential of PopuCATH to prevent bacterial infection.

Systematic computer analysis of published literature on nutritional support for vaccination

A range of 6700 publications from the PubMed database on the association of micronutrient supply and results of antibacterial and antiviral vaccination was reviewed by the method of topologic and metric analysis. This method allows for a selection of features (i.e. key words) by their informativity, the establishment of the most informative that provide the basis for “synthetic” features and algorithms, or the classification of the reviewed text by the relevance to the subject of the study. The results of fundamental studies showed that folates, vitamins A, D, and B12 are the regulators of mitosis of T and B-lymphocytes that exert the functions of the acquired immunity. Such microelements as zinc, iron, selenium, manganese, and omega-3 polyunsaturated fatty acid support the functioning of T and B-lymphocytes (energy metabolism, intracellular signal transmission, and transcription). Clinical studies showed that the support of vaccination with the specified micronutrients not only increases the titre of the respective antibodies to viral and bacterial pathogens but can also prevent unfavorable effects from vaccination. The administration of micronutrients before and after vaccination will contribute to a decrease in the mortality rate and severity of the pathology development (in case of disease). A systematic analysis allowed the authors to determine the perspectives of the proposed measures for an increase in the effectiveness and safety of vaccines, including COVID-19. Additional micronutrient supply contributes to an increase in the effectiveness and safety of vaccination. The application of specialized vitamin and mineral complexes during vaccination is economically feasible and reduces the vaccination risks for patients with polyhypoavitaminoses.

Immunomodulatory effect of the combined use of Vetosporin Zh probiotic and Gumi-malysh biologically active additive

Background and Aim: Various means and methods, including probiotics and biologically active additives, have been developed and proposed for production to increase the immunobiological reactivity of the body, regardless of the etiology of its decrease. This study aimed to find out the immune status of calves during the preweaning period in association with Vetosporin Zh, Normosil, and Gumi-malysh. Materials and Methods: The research object was 30-day-old calves of black-and-white Holstein breed. The calves were divided into four groups of 20 heads each. The calves of the first, second, and third experimental groups were treated with Normosil probiotic, Vetosporin Zh probiotic, and Vetosporin Zh probiotic in combination with Gumi-malysh, respectively. The calves in the first, second, and third experimental groups were treated with Normosil probiotic, Vetosporin Zh probiotic, and Vetosporin Zh probiotic in combination with Gumi-malysh, respectively. Results: On days 10 and 21 of the experiment, animal blood was collected to determine the content of total protein, protein fractions, immunoglobulins, T and B lymphocytes, phagocytic activity and a phagocytic number of neutrophils, and circulating immune complexes (CIC). The combined use of Vetosporin Zh probiotic (dose, 20 mL) with Gumi-malysh (dose, 30 mL) per animal for 30 days in 1-month-old calves contributes to the increase in the number of T lymphocytes, B lymphocytes, and immunoglobulin A (IgA) and immunoglobulin G (IgG) levels by 2.9%, 3.8%, and 0.96 and 2 g/L, respectively, while reducing the immunoglobulin M (IgM) level; an increase in the phagocytic activity of blood neutrophils and the phagocytic number by 7% and 1.8%, respectively, as well as a decrease in the CIC level with similar indicators in calves that were not treated with the agents. Conclusion: The method used in the current study helps increase the number of T and B lymphocytes, increase IgA and IgG levels while reducing IgM levels, and increase the phagocytic activity and a phagocytic number of blood neutrophils, as well as decrease the CIC level.

Assembly of a spatial circuit of T-bet–expressing T and B lymphocytes is required for antiviral humoral immunity

Effective antiviral immunity requires generation of T and B lymphocytes expressing the transcription factor T-bet, a regulator of type 1 inflammatory responses. Using T-bet expression as an endogenous marker for cells participating in a type 1 response, we report coordinated interactions of T-bet–expressing T and B lymphocytes on the basis of their dynamic colocalization at the T cell zone and B follicle boundary (T-B boundary) and germinal centers (GCs) during lung influenza infection. We demonstrate that the assembly of this circuit takes place in distinct anatomical niches within the draining lymph node, guided by CXCR3 that enables positioning of TH1 cells at the T-B boundary. The encounter of B and TH1 cells at the T-B boundary enables IFN-γ produced by the latter to induce IgG2c class switching. Within GCs, T-bet+ TFH cells represent a specialized stable sublineage required for GC growth but dispensable for IgG2c class switching. Our studies show that during respiratory viral infection, T-bet–expressing T and B lymphocytes form a circuit assembled in a spatiotemporally controlled manner that acts as a functional unit enabling a robust and coherent humoral response tailored for optimal antiviral immunity.

T- and B-lymphocytes subpopulations in the early and advanced rheumatoid arthritis

Objective: to evaluate changes in T- and B-lymphocyte subpopulations at different stages of rheumatoid arthritis (RA).Patients and methods. The study included 53 patients with a definite RA diagnosis according to the 2010 ACR/EULAR criteria (mean age 54.2 [47; 62] years). Group 1 included 27 patients (25 women and 2 men) without history of synthetic disease modifying anti-rheumatic drugs (sDMARDs) intake, group 2 included 26 patients (22 women and 4 men) receiving sDMARDs (methotrexate or leflunomide). The control group consisted of 29 healthy volunteers (23 women and 6 men), the median age was 58.5 [53; 62] years. In all participants flow cytofluorometry according to the standard technique with immunophenotyping of T- and B-lymphocytes was performed.Results and discussion. Compared to controls, patients in group 1 who had not previously received sDMARDs showed a transient increase in "switched" memory B-cells, transient B-cells, and plasmablasts, which was not observed in patients of group 2 (on sDMARDs therapy). Patients with advanced RA showed a statistically significant decrease in the absolute and relative number of memory B-cells, the absolute and relative number of "switched" B-lymphocytes, as well as the number of plasmablasts and transient cells. In RA patients, a statistically significant rela tionship was established between the number of swollen joints and the level of plasmablasts (r=0.51), memory cells (r=0.54), and "switched" B-cells (r=0.41), p< 0,05 in all cases. There were no statistically significant changes in other subpopulations of B-lymphocytes and the profile of T-lymphocytes.Conclusion. Changes in the B-lymphocyte profile are characteristic of different stages of RA. At an early stage, there is an increase in the number of transient B-lymphocytes, plasmablasts and plasmocytes, and in the advanced stage, a decrease in the level of certain populations of B-lymphocytes, such as memory B-cells and "switched" B-lymphocytes. It can be assumed that the ineffectiveness of sDMARDs is associated with a change in the population composition of B-lymphocytes, which requires further study.

The condition of piglet immune system during post-weaning period depending on the sex and stress-reactivity

The immune system of 90-day old piglets of SM-1 Novosibirsk breed piglets depends on sex and stress-reactivity. Stress-reactivity was measured using halothane test. The immunologic testing was performed 30 days after weaning. Our results show that overall piglet immune system demonstrated high proliferative activity of T- and B- immunocompetent cells with active formation of mature active T-and B-lymphocytes and did not show signs of immunosuppression. Compared to guilts, barrows had higher concentration of leucocytes, T-and B-lymphocytes, killer-supressor T-cells, activated and poorly differentiated T-lymphocytes. Gilts had higher production of inductor-helper T-cells, IgM and IgG when compared to barrows. Stress-resistant piglets had higher concentration of B-lymphocytes, IgM and IgG whereas stress-sensitive piglets had higher concentration of T-lymphocytes, supressor-killer T-cells and thymus T-lymphocytes. Gilts had higher concentration of inductor-helper T-cells than killer-supressor T-cells. Gilts overall had intensive antibody synthesis, however, stress-resistant gilts had higher IgG synthesis compared to stress-sensitive gilts. In barrows immature T-lymphocytes differentiated mainly into killer-supressor T-cells. The adaptivity of barrow immune system was characterized by high circulatory B-lymphocytes and IgM. Stress-sensitive barrows had lower antibody synthesis levels and higher T-lymphophoesis compared to stress-resistant barrows.