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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.

Resting energy expenditure (REE) by indirect calorimetry and body composition by bioimpedance analysis are studied in three groups of children aged 5–18 years. Group 1 (n = 181) – patients in remission of cancer, group 2 (n = 55) – children with oncology diseases receiving chemotherapy or who are in the early period after hematopoietic stem cell transplantation, group 3 (n = 63) – children with non-malignant diseases of the gastrointestinal tract. To eliminate the influence of age and gender on the intergroup comparisons, body composition parameters were expressed as standardized values (z-scores) relative to a reference group of healthy Russian children (n = 138,191). Group 1 was characterized by excess fat content with intact lean body mass, and groups 2 and 3 by protein depletion, more pronounced in Group 2 with a higher percentage of body fat. All used conventional formulas (WHO, Harris–Benedict and others) in groups 1 and 3 underestimated REE as compared with indirect calorimetry. A new formula for REE, giving an unbiased estimate in the group 1 was proposed: REE (kcal/day) = 28.7 × BCM (kg) +10.5 × Height (cm) – 38.6 × Age (years) – 134, where BCM – body cell mass according to bioimpedance analysis (R2 = 0.67, the standard deviation of 196 kcal/day).

Hematopoietic stem cells transplantation (HSCT) is currently widely used for solid tumors, leukemia and autoimmune diseases therapy. Complications in post-transplant period, as well as specific therapy of these complications lead to nutritional status changes, which worsen post-transplant period and influence of outcome. To analyze nutritional status and tissue composition characteristics and determine the value of bioimpedance methods for complex examination of nutritional status in children after HSCT are the goal of this study. Changes in nutritional status and tissue composition of 34 children during HSCT due to various malignant and non-malignant diseases were analyzed. Bioimpedance measurements and somatometry was used to assess tissue composition. Significant worsening of nutritional status, skeletal muscle mass loss with a relatively stable adipose tissue to day +100 has been shown. A significant decrease of active cell mass and phase angle in all study phases were revealed. A high correlation between body fat mass index and skin-fat folds size over the triceps (r = 0.86), as well as between skeletal muscle mass index and shoulder muscles circle size (r = 0.82) was revealed. It is concluded that in the early post-transplant period (up to 100 days) nutritional status significantly worsened and tissue imbalance developed with a relative predominance of fat component and somatic protein pool reduction. Importance of comprehensive evaluation and monitoring of nutritional status to develop a strategy for nutritional support in children after HSCT is discussed.

The results of our cross-sectional bioimpedance study of children aged 7–17 years cured of cancer during follow-up (patients’ group, n = 552, remission time range 0–15 years) and of age-matched healthy controls (n = 1,500) show significant intergroup differences in body height and body composition parameters. The most pronounced alterations in the patients’ group were observed in standardized values of phase angle reflecting a sharp decrease in the percentage of metabolically active body cell mass in fat-free mass. Malnutrition, judged from the prevalence of obesity and low phase angle, was observed in 52.7 % of our patients reaching a maximum of 76.8 % in a subgroup of children with CNS tumors. In view of known association that exists between malnutrition and reduced tolerance to chemotherapy, increased susceptibility to infections and adverse outcomes rate, we recommend using bioimpedance analysis in remission of childhood cancers in order to monitoring and timely correction of nutritional state as well as for prevention of delayed cardiovascular risks.

Hematopoietic stem cell transplantation (HSCT) is widely used in the treatment of malignant and autoimmune diseases. Various complications often develop during the post-transplantation period that can significantly impair the clinical outcomes, so the ability to predict therisk of severe complications is of great practical importance. Predictive value of some anthropometric indices and bioimpedance analysis(BIA) measured before conditioning to assess the risks of serious complications and graft hypofunction in the early post-transplant period(100 days) were analyzed. Anthropometry and BIA used in a comprehensive assessment of nutritional status in order to optimize the nutritional support of these patients. 101 patients were examined before conditioning and at different times during the early post-transplant period: 50 children (5–17 years of age) were examined using BIA and anthropometry, 61 children (6 months – 4 years of age) – using only anthropometry without BIA due to age restrictions. The prognostic value of the phase angle (FA), ratio of the active cell mass to lean body mass (ACM/LBM) and shoulder muscle circumference (SMC) was shown. Thus, in patients with FA ≤ 4, ACM/LBM < 0.45 and SMC ≤ 10th percentile before conditioning risk of severe complications during early post-transplant period was significantly higher (p < 0.05). Also, in patients with FA ≤ 4 and ACM/LBM < 0.45 a significantly higher risk of graft hypofunction developing was observed (p < 0.05).

The aim. To estimate heat capacity (C) measurements for individual dietary support of pediatric cancer patients during chemotherapy. Material and methods. 43 cancer children are examined. Group 1: 15 children, 5 children receiving polychemotherapy, 10 children – in post-BMT period (5 of them were re-examined on clinical indications). Thus we got 21 resting heat capacity measures. Group 2 includes 14 children with cancer after completing chemotherapy. Group 3 (control group) – 14 children with gastroenterological pathology. (C) is determined by indirect calorimetry, then basal metabolic rate (BMR) is calculated by formula BMR=90%C and compared with (Cf), calculated by formula WHO (1985). The results. In group 1 the average values of BMR and Еоф considerably differ: 986,5 and 1285,9 kcal accordingly (p

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.