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Animal tumor models are used for preclinical studies of drugs and cancer therapy. The aim of this work was to analyze the growth of murine pancreatic tumor cells Pan02, carrying GFP marker, injected subcutaneously (s. c.), intraperitoneally (i. p.) or orthotopically into the pancreas (ortho) of C57BL/6 mice. Mice were injected with 2 × 105 cells: s. c. in the right flank; i. p. with a syringe into the abdominal cavity, or ortho surgically under the pancreas capsule. The weight of mice was determined in the dynamics of tumor growth, and blood serum was taken to analyze the antibody response to the GFP reference protein. At the 2nd and 4th weeks of tumor growth, some mice were slaughtered and the expression of GFP by the tumor cells, as well as the composition of the immune cells in the tumor, were analyzed by flow cytometry and confocal microscopy. It was shown that with the different localization, the pancreatic tumors grew at different rates and lethality. When the tumor was injected i. p., mice lost weight with rapid tumor growth. In the ortho model, the mice increased their weight. Mortality in the s. c. and i. p. groups was comparable. In the s. c. model, the tumor grew slowly to a volume of 200-400 mm3 and stopped growing. There was no mortality in this group during the follow-up period (2 months). The same antibody response to GFP was formed with all injection schemes. The subpopulation composition of immune cells varied greatly in the different models of tumor cell administration. Regardless of the type of immune response, Pan02-GFP cells rapidly suppressed GFP gene expression in vivo. The data obtained showed that murine pancreatic tumor Pan02 is immunogenic and causes the formation of an adaptive immune response. Regardless of the presence or absence of an immune response and elimination of GFP+ cells, the tumor continued to grow in the i. p. and ortho models, but not in the s. c. one, and caused the death of mice. When conducting preclinical studies, it is necessary to use several ways of tumor cell injection to obtain a more objective result.

Type I allergy is mediated by the formation of IgE antibodies to proteins secreted by nonreplicating microorganisms (plant pollen, house dust mites, etc.) that enter the mucous membranes in very low concentrations. The mechanisms and localization of naive B cells’ switching to IgE production have not been fully determined. The aim of this work was to determine the switching site of B cells and the traffic of IgEproducing B cells in mice immunized with a low dose of equimolar mixture of egg proteins Gal d1, Gal d2, and Gal d3. Allergens in saline solution were injected into the withers of mice 9-10 times with an interval of 2-3 days; the total dose was 2.7 µg/mouse. The production of IgE to Gal d proteins in the blood and by B cells isolated from the withers, draining lymph nodes, spleen, and bone marrow of immune mice was analyzed in dynamics after cessation of sensitization. Both in blood and in in vitro cultures, the dominance of IgE changed from the recognition of the LMW Gal d2 during the sensitization of mice to the HMW Gal d3 after sensitization was discontinued. In this model, an IgG memory response appeared only a month after the end of sensitization and recognized only Gal d3. In vitro cultures showed that B cells switched to IgE production locally in the withers with low traffic to the spleen. In the blood serum, IgE titers for all Gal d proteins decreased after the cessation of sensitization and persisted for a long time. A month after the cancellation of the sensitization, a pool of B cells producing IgE in vitro appeared in the spleen. These B-cells died after 20-30 days as no in vitro IgE production was observed later than 85-90 days. The results obtained allowed us to draw several conclusions. B cells switch to IgE synthesis locally at the site of allergen injections. The response was two-phase: LMW Gal d2 was recognized in the early response, while HMW Gal d3 was recognized in the late phase. In this model, the IgG response to HMW Gal d3 was clearly dominant. In conclusion, it has been shown that when the immune system recognizes a mixture of proteins originating from some allergen, the dominance of proteins recognized by both IgE and IgG is observed. Since allergy patients most often do not have IgG antibodies, it can be assumed that in this case an acute phase response, supported by antigen intake, is observed, in which LMW allergens are recognized.

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.

In view of increased incidence of IgE-mediated diseases in Russia and worldwide, the efforts of various research groups are focused on studying the mechanisms that trigger the process of switching B-lymphocytes to IgE synthesis upon human exposure to harmless allergens, including the role of various antigen-presenting cells (APCs) in this process. However, the role of distinct APCs upon long-term penetration of low antigen doses via the tissue barriers, is yet poorly understood, as well as specific features of these events upon entrance of the antigen through the subcutaneous adipose tissue which contains tissueassociated lymphoid clusters (TALC).The aim of this work was to determine the relationship between the local accumulation of various APCs in the subcutaneous adipose tissue and development of early IgE production in a clinically relevant experimental model of allergy with long-term administration of low allergen doses. In this experimental model, specific IgE synthesis is induced, with minimal concomitant IgG production, thus mimicking the situation observed in patients with clinically sound allergies. BALB/c mice were immunized for 4 weeks subcutaneously in the withers area or intraperitoneally with low (100 ng) or high (10 μg) doses of the model allergen (ovalbumin). Blood samples were taken weekly from mice for ELISA testing, to determine the production of specific antibodies. Provocation tests were performed with high dose of the allergen, and adipose tissue samples were taken from the site of antigen injection for flow cytometric assays, in order to evaluate the contents of various APC subpopulations. Specific IgE production was induced mainly by subcutaneous injection of the antigen at low doses (100 ng) into the area of withers. When using this experimental regimen, we observed accumulation of classical CD11b+ cells in adipose tissue at the withers site, but not in the peritoneal adipose tissue, in absence of CD11b- classical, inflammatory or plasmacytoid, dendritic cells. These findings coincided in time with increased production of specific IgE on days +14 to +21. Accumulation of CD11b+CD11c- macrophages and their CD206+ M2 subpopulations at early terms (days +7 and +21) was also observed only after subcutaneous injection of the antigen into the withers area. The high-dose antigen injection (10 μg) which mediated IgG1 production to greater extent than production of IgE, led to earlier accumulation of CD11b+ classical dendritic cells (on day 7th), and to the absence of macrophage accumulation at later stages (day 21th). Thus, the early start of specific IgE production upon low-dose injection of the antigen into the subcutaneous adipose tissue may be associated with its presentation by CD11b+ classical dendritic cells in the presence of CD11b+CD11c- macrophages.

Antigen-binding fragments of antibodies specific to the tumor-associated ganglioside GD2 are well poised to play a substantial role in modern GD2-targeted cancer therapies, however, rapid elimination from the body and reduced affinity compared to full-length antibodies limit their therapeutic potential. In this study, scFv fragments of GD2-specific antibodies 14.18 were produced in a mammalian expression system that specifically bind to ganglioside GD2, followed by site-directed pegylation to generate mono-, di-, and tetra-scFv fragments. Fractionated pegylated dimers and tetramers of scFv fragments showed significant increase of the binding to GD2 which was not accompanied by cross-reactivity with other gangliosides. Pegylated multimeric di-scFvs and tetra-scFvs exhibited cytotoxic effects in GD2-positive tumor cells, while their circulation time in blood significantly increased compared with monomeric antibody fragments. We also demonstrated a more efficient tumor uptake of the multimers in a syngeneic GD2-positive mouse cancer model. The findings of this study provide the rationale for improving therapeutic characteristics of GD2-specific antibody fragments by multimerization and propose a strategy to generate such molecules. On the basis of multimeric antibody fragments, bispecific antibodies and conjugates with cytotoxic drugs or radioactive isotopes may be developed that will possess improved pharmacokinetic and pharmacodynamic properties.