Cellular Composition Of Peripheral Blood Research Articles

Single Cell Multi-Omic Dissection of Response and Resistance to Chimeric Antigen Receptor T Cells Against BCMA in Relapsed Multiple Myeloma

Background: Chimeric antigen receptor (CAR) T cell therapy has revolutionized treatment of relapsed/refractory multiple myeloma (RRMM). Robust variables that predict long-term response are currently missing. Limited data are especially available on the impact of bridging therapies on manufacturing and outcome. We conducted a longitudinal single-cell multi-omics study to identify factors that predict response to BCMA-directed CAR T cells. Changes in the immune microenvironment associated with response were analyzed as well as the impact of prior bridging therapy with bispecific antibodies on subsequent CAR T cell manufacturing and outcome. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 29 consecutive MM patients treated with commercially available anti-BCMA CAR T cells on the day of leukapheresis as well as days 30 and 100 after CAR T cell infusion. PBMCs were subjected to single cell RNA, T-cell receptor (TCR) and B-cell receptor (BCR) sequencing. A custom panel of 57 oligonucleotide-coupled antibodies was used to study surface proteomics. Downstream analyses were performed with Seurat. Differences in cellular compositions at all three time points were analyzed with scCODA. To analyze CAR T cell functionality, CAR T cells from peripheral blood were isolated 7 days after infusion and subjected to an in vitro cytotoxicity assay after expansion and stimulation. Patients were grouped based on their best response following CAR T cell infusion (CR: n=12, non CR: n=17). Results: In total, 375,338 cells were sequenced (median 7246 cells/sample, range 1,569-10,972 cells) and 354,878 cells (94.5%) passed quality assessment. Quantitative and qualitative differences in the cellular composition of peripheral blood between CR and non CR patients were detected at the time of leukapheresis as well as on days 30 and 100 following infusion. CR patients harbored significantly more CD8+ effector memory T cells (TEM) at leukapheresis and less NK cells on day 30 after therapy compared to non CR patients. Regulatory T cells isolated at the time of leukapheresis from non CR patients exhibited significantly higher surface protein levels of CXCR3, CD40, CD95 and KLRG1 (p<0.015, respectively) that have been associated with T cell senescence and impaired tumor immunity. No significant differences in cell numbers between CR and non CR were detected on day 100 after CAR T cell infusion. However, single cell TCR analysis revealed an increasing diversity in the TCR repertoire over time in patients with CR, while Shannon diversity decreased from leukapheresis over day 30 to day 100 in non CR patients (p=0.004). The prior administration of the bispecific antibody teclistamab had no significant impact on the quantitative cellular composition at the time of leukapheresis. However, termination of manufacturing in the first attempt occurred in all patients with a close proximity of teclistimab administration and apheresis. Differential gene expression analysis showed that the application of teclistamab was associated with impaired T cell activation and exhaustion indicated by upregulation of e.g. CTLA4, TIGIT, LAG3 and GZMK. After discontinuation of teclistamab (median 4 weeks) and successful manufacturing of CAR T cells, we found no significant differences for in vitro cytotoxicity and in vivo expansion of CAR T cells. CAR T cells isolated at day 7 post-infusion from patients in CR, non CR or with prior teclistamab exposure, effectively eliminated MM cells (U-266). Tracking of single CAR T cells over time showed that the majority of CAR+ cells were CD8+ TEMs regardless of remission achievement or prior teclistamab exposure. Conclusion: We demonstrate that differences between MM patients achieving a CR and patients with suboptimal response upon anti-BCMA CAR T cell therapy can already be identified at the time of leukapheresis. Long-term changes associated with CR include a diversification of the TCR repertoire. Successful CAR T cell manufacturing is hampered by exposure to bispecific antibodies but can be successfully achieved by allowing for a wash-out phase of ca. 4 weeks.

The Impact of Lymphocyte Composition in the Stem Cell Harvest on Blood Lymphocytes 1-2 Weeks Post-Autologous Stem Cell Transplant for Newly Diagnosed Myeloma

Background It is known that cellular composition of the stem cell harvest in autologous stem cell transplantation for multiple myeloma is important for engraftment and blood reconstitution, with CD34 count commonly used as a marker for harvest adequacy; however, there is a relative lack of data on the importance of lymphocyte marker-expressing cells in the harvest and their implications for immune reconstitution and outcome. Here, we demonstrate that the amount and proportion of lymphocyte marker-expressing cells in the stem cell harvest directly correlate with the cellular composition of the peripheral blood 1-2 weeks post-transplant. Objective To investigate whether the immunological cellular composition of the stem cell harvest prior to autologous stem cell transplantation in multiple myeloma correlates with the cellular composition of peripheral blood 1-2 weeks post-transplant. Methods We analysed stem cell harvest (SCH) and post-autologous stem cell transplant (PASCT) cellular composition data for 185 patients from the Myeloma XI trial. Utilising flow cytometry, SCH data was obtained from a 0.5ml aliquot of the total harvest and PASCT data was obtained from a peripheral blood sample collected 1-2 weeks post-transplant. Spearman's rank tests were performed across a variety of cell markers to assess for correlations between the cell count per µl and for correlations between the cell percentage (defined as the proportion of total CD45+ cells in each sample that expressed the given combination of cell markers) between the SCH and PASCT samples. Analysed cell marker phenotypes included CD45+, total CD3+, CD3/4+, CD3/8+, CD19+, and CD16/56+. Results We demonstrated statistically significant correlations for multiple cell markers between the composition of the SCH and peripheral blood 1-2 weeks post-transplant. Regarding cell count per µl, significant correlations were found in CD45+ (r=0.2168; p=0.0030), CD3+ (r=0.2254, p=0.0020), CD3/4+ (r=0.2050, p=0.0051), CD3/8+ (r=0.2733, p=0.0002), CD19+ (r=0.2787, p=0.0001), and CD16/56+ (r=0.3085, p<0.0001). Regarding cell percentages, significant correlations were found in CD3+ (r=0.3013, p<0.0001), CD3/4+ (r=0.3551, p<0.0001), CD3/8+ (r=0.4640, p <0.0001), CD19+ (r=0.2080, p=0.0048), and CD16/56+ (r=0.3221, p<0.0001). Further data assessing for correlations between harvest composition and progression-free and overall survival is also being analysed. Conclusions These data demonstrate clear correlations between higher lymphocyte counts in the stem cell harvest and higher lymphocyte counts 1-2 weeks post-autologous stem cell transplantation. Additionally, correlations between cell percentages indicate that harvest composition is directly correlated to the cellular composition of the blood following transplant. The correlations were statistically significant for a variety of phenotypes, including T cells, B cells, and NK cells, and indicate that the total dosage of lymphocytes re-introduced during transplantation may have wide ramifications for immune reconstitution as early as 1-2 weeks post-transplant. Our findings demonstrate that cellular composition of the harvest is a potentially important consideration that could influence immune reconstitution following autologous transplantation for myeloma. This research suggests that the lymphocyte constitution of the stem cell harvest, currently an overlooked metric during harvest collection, is correlated with immune recovery and further investigations may reveal implications for transplant outcome.

Covid-19 and ischemic heart disease: clinical manifestations, biomarkers, treatment and follow-up

Funding AcknowledgementsType of funding sources: None. The aim of the study: To research the correlation between in-hospital mortality and markers of systemic inflammation in patients with COVID-19 with Ischemic heart disease (IHD) and to evaluate hematological indices under the influence of using quercetin and arginine in basic therapy.Material and methodsThe data from 52 case histories of patients with COVID-19 and IHD, who were hospitalized from February till April 2021, has been analyzed. 40 patients who recovered and were discharged from the hospital were included to group I, 12 patients who died were included to group II. The evaluation of hematological indices under the influence of adding metabolic drugs in the complex therapy was determined in 27 patients from group I, which was also divided into 2 subgroups. Subgroup I A, which consisted of 17 patients, received water-soluble quercetin and subgroup I B (10 patients) - arginine hydrochloride. The severity of systemic inflammation syndrome in the selected groups of patients was evaluate by the number of leukocytes and the cellular composition of peripheral blood on admission of patients to the hospital and on the basis of obtained data the values of hematological integral indices were calculated: blood leukocyte shift index (BLSI) according to N.I. Yabluchansky, neutrophil-lymphocyte ratio (NLR), leukocyte index of intoxication (LII) was determined by J. Kalf-Kalif, nuclear index by G.A. Dashtayantz, neutrophil-to-monocyte ratio (NMR), lymphocyte-to-eosinophil ratio (LER), lymphocyte-to-monocyte ratio (LMR), allergy index (AI).ResultsPatients from group II, who died of cardiovascular complications associated with COVID-19, had a significantly higher level of systemic inflammatory response, which is manifested by a significant increase of the total number of white blood cells (16,85±5,25) × 109/L, in contrast to patients of group I, who were discharged with recovery (5,81±1,75) × 109/L (p<0,05), and had the increasing number of neutrophil percentage (88,09±2,99) % against to (67,71±9,85) % (p<0,05), with a decrease in the percentage of peripheral blood lymphocytes (6,25±1,42) % in patients from group II against (18,13±4,25) % in patients of group I. The patients of clinical group II had the increasing of a number of hematological indices - BLSI: (9,28±2,16) against group I (2,47±0,95) (р<0,05), NLR: (26,99±11,11) against (4,01±2,36) and also the increase of NMR in group II (14,81±3,49) comparing to group I (6,22±2,18) (р<0,05). The obtained results analysis of the distribution of hematological indices in patients of subgroups I A and I B after adding quercetin and arginine to the basic therapy in the inpatient phase of treatment showed that a significant difference between the indicators is not observed.ConclusionsThe results of the study not only suggest new markers of mortality from severe COVID-19, but also identify potential ways of treatments.

ВОЗДЕЙСТВИЕ БОЛЕВОГО СТРЕССА НА АДАПТИВНЫЕ ВОЗМОЖНОСТИ СИСТЕМЫ КРОВИ И БЛАГОПОЛУЧИЕ ЛОШАДЕЙ

The full genetic potential of productive animals can only be realised if high standards of welfare are maintained in production. One of the indicators of animal welfare is animal health, and one of the markers of various pathological processes is pain. Sports horse breeding is a specific concept in which the competent work of trainers and riders significantly effects on the health of animals, and therefore their performance, in addition to housing and feeding conditions. Timely diagnosis of pain is important for evaluating animal welfare, as pain can be considered as a predictor of various diseases. Using the standardized Horse Grimace Scale (HGS) pain scale, the population of sports horses of the equestrian sports complex of Russian State Agrarian University – Moscow Timiryazev Agricultural Academy was evaluated for the presence and intensity of pain syndrome, followed by analysis of some blood parameters. A correlation has been established between the intensity of pain and the number of diseases previously diagnosed in animals. Pain, as a powerful stress factor, causes tension in almost all functional systems of the body, including the blood system. Horses with a severe pain syndrome showed changes in the cellular composition of peripheral blood, in particular, an increase in the level of leukocytes, a decrease in the content of erythrocytes and hemoglobin content therein, with a simultaneous increase in the mean erythrocyte volume and mean corpuscular haemoglobin,, as well as an increase in glucose concentration. Such changes can be considered as adaptive mechanisms that make the animal body function normally under the effect of pain stress.

The connection between hospital mortality and inflamation markers in COVID-19 patients and ischaemic heart disease

The purpose of the work to investigate the connection between hospital mortality and markers of systemic inflammation in COVID-19 patients with ischemic heart disease (IHD).Material and methods. The data of 52 patients’cases of diseases on COVID-19 with IHD who underwent inpatient treatment were analyzed. The first group included 40 patients who were discharged with recovery, and the second group included 12 patients who died. The degree of systemic inflammation syndrome in the selected groups of patients was estimated by the number of leukocytes and cellular composition of peripheral blood upon admission to the hospital, and based on the data received, the value of hematological integral indices was calculated.Results. It has been established that group II patients who died of cardiovascular complications connected with COVID-19 had significantly higher levels of systemic inflammatory response, which exhibits a significant increase in the total blood white blood cell content and increases in the percentage of neutrophils with a decrease in the percentage of lymphocytes and is substantially higher than the integrated haematological indices: leukocyte shift index, index of the ratio of neutrophils to lymphocytes and index of the ratio of neutrophils to monocytes. During the systemic inflammatory reaction, it has been observed that the inflammatory process aggravates the coronary atherosclerotic plaque making them more susceptible to rupture. Therefore, we can assume that existing IHD combined with an enhanced inflammatory response can lead to heart damage in patients infected with SARS-CoV- 2.Conclusion. Lymphocytopenia, excessive activation of the inflammatory cascade and heart damage are important features of COVID-19 disease and have high predictive value.

Comprehensive assessment of the systemic and local immunity, inflammatory factors and clinical indicators in patients with gastric cancer

Objective.To study the state of cellular immunity and local immunity in patients with gastric adenocarcinoma.Materials and methods.From 2017 to 2018 45 primary patients with gastric adenocarcinoma (25 — with stages I–III, 20 — with stage IV) received surgical / combined treatment or chemotherapy at the Blokhin Scientific Research Center of Oncology, respectively. Peripheral blood and tumor tissue were collected before starting treatment. The percentage of the degree of infiltration of tumor tissue by lymphocytes (CD45 + CD14‑TILs) was assessed by flow cytometry: T‑cells (CD3 + CD19‑TILs), B‑cells (CD3‑CD19 + TILs), NK‑cells (CD3‑CD16 + CD56 + TILs), effector cells CD16 and CD8 and their cytotoxic potential (CD16 + Perforin + TILs, CD16CTPTILs), (CD8 + Perforin + TILs; CD8CTPTILs); subpopulations of regulatory T‑cells — NKT‑cells (CD3 + CD16 + CD56 + TILs), regulatory CD4‑cells (CD4 + CD25 + CD127‑TILs) and CD8 (CD8 + CD11b‑CD28‑TILs) and parameters of systemic immunity.Intratumoral and stromal subpopulations of CD4 + TILs, CD8 + TILs, CD4 + / CD8 + TILs ratios were studied by immunohistochemistry. Also, the cellular composition of peripheral blood was investigated. The prognostic significance of immune cells, inflammation factors (neutrophil‑lymphocyte index, platelet‑lymphocyte index) and clinical characteristics (patient»s age (both by years and by groups: up to 45 years, 46–60 years, over 60 years), disease stage, differentiation (G), Lauren type and MSI status were evaluated for overall survival (OS) and progression‑free survival (PFS).Results.The factor of a favorable prognosis for PFS in patients with local and locally advanced forms of gastric cancer was an increase in the number of CD3 + CD19‑TILs (HR0.865, 95 %CI 0.782–0.957, p = 0.005), and for poor prognosis — an increase in NK‑cells; HR1.382, 95 %CI 1.087–1.758, p = 0.008. There was a negative effect of the relative content of NK‑cells, an increase in the level of neutrophils in the peripheral blood on the OS of patients with metastatic GC (HR1.42, 95 %CI 1.06–1.89, p = 0.017 and HR1.64, 95 %CI 1.12–2.40, p = 0.011). At the same time, an increase in the age of patients, the level of neutrophils and platelets (HR1.106, 95 %CI 1.002–1.199, p = 0.015; HR1.714, 95 %CI 1.063–2.764, p = 0.027 and HR1.017, 95 %CI 1.006–1.029, p = 0.003) reduce PFS in patients with metastatic gastric cancer.Conclusion.Indicators of local immunity, the cellular composition of peripheral blood, characterizing the systemic inflammatory response, as well as indicators of systemic immunity can serve as additional prognostic factors in gastric cancer.

ХАРАКТЕРИСТИКА ИММУННОГО СТАТУСА ЮНЫХ ХОККЕИСТОВ ПУБЕРТАТНОГО ВОЗРАСТА

Relevance. In modern publications, sufficient attention is paid to the study of the cellular composition of peripheral blood and the immune status of athletes. These studies are carried out at various stages of the training process in order to establish a connection between the registered changes and the nature and severity of muscle loads. The available literature data mainly relate to the indicators of the immunity of adult athletes involved in various sports, recorded at various stages of training and characterizing the adaptation to muscular load. The state of the immune system largely determines the success of a sports career in modern sports. Aim of the study: to assess the immune profile (cellular and humoral link) of pubertal hockey players at the beginning of the preparatory stage of the training period. Materials and methods. To assess the immune profile (cellular and humoral link) of pubertal hockey players at the beginning of the preparatory stage of the training period, the relative and absolute content of lymphocytes was studied by the method of immunophenotyping with the determination of their subpopulation composition (CD3 +, CD3 + CD4 +, CD3 + CD8 +; CD3- CD16 + CD56 +, CD19 +, CD4 + / CD8 +), and by turbidimetry - specific proteins (immunoglobulins A, G, M). Results. The obtained values of the studied indicators did not differ from those of adolescents who do not go in for sports, and generally fit into the age norms. Isolated cases of deviations are associated with the characteristics of the morbidity of the examined adolescents. The recorded immune profile of athletes will be used as reference values during the continuation of the study (at the end of the pre-competition and post-competition periods), the purpose of which is to monitor the impact of the training process on the immunity of athletes of puberty. The obtained values of the studied indicators do not differ from those in the control and, fit into the age norms. In conclusion, the authors come to the conclusion that it is possible to use the recorded immune profile of athletes as reference values during the continuation of the study (at the end of the preparatory stage and at the end of the competitive period), the purpose of which will be to assess the impact of the training process on the immunity of athletes of puberty.

STUDY OF THE GENERAL TOXIC PROPERTIES OF THE ANTI-TB DRUG THIOSONIDE

The aim of the study was to evaluate the subchronic and chronic toxicity of the substance – thiosonide - in experiments on rabbits and rats. The animals received doses ranging from therapeutic to 75 times higher. The drug in studied doses did not cause any significant changes in body weight increase of animals, behavioral reactions, food and water consumption. There were no changes in the biochemical and cellular composition of peripheral blood, the protein-synthesizing function of the liver, carbohydrate metabolism, and the functional state of the cardiovascular system. In the work of the central nervous system, a slight decrease in motor activity was observed in male rats that were long-term treated with a drug substance in a dose of 100 mg/kg (more than 10 times higher than the therapeutic dose). A local irritant effect in the gastrointestinal tract of experimental rats was noted according to the results of histological studies, most pronounced under the influence of the drug at a dose of 100 mg/kg. According to the results of histological studies, minor dystrophic changes in the liver, kidneys and myocardium were found in some animals under the influence of a drug substance at a dose of 100 mg/kg. Thus, the active substance of the anti-tuberculosis drug does not have toxic properties that limit its long-term use, which allows us to consider it as a promising tool for inclusion in the basic long-term courses of treatment of the disease.

Pathogenetic mechanisms for the development of hematological disorders in induced fatty liver disease in Wistar rats and assessment of the regulatory effects of carnosine and α-lipoic acid

The study of the relationship between hematopoiesis and metabolism is now particularly relevant in view of the high incidence of alimentary dependent diseases, including non-alcoholic fatty liver disease. In this regard, pathogenetic factors of this disease development are studied actively in order to choose adequate drug therapy and usage of bioactive substances with antioxidant properties. The aim of the study was to study the pathogenetic relationship of hematological disorders and imbalance of growth factors, leptin and ghrelin in male Wistar rats in the model of the initial stage of non-alcoholic fatty liver disease development and to assess the regulatory effect of minor bioactive substances - carnosine and α-lipoic acid. Material and methods. The studies were performed on male Wistar rats with initial body weight 150±10 g within 8 weeks. Animals were divided into 5 groups (n=8 in each). Rats of the control group received a complete modified diet AIN93M, in which soybean oil was replaced with sunflower oil and lard (1:1). Rats of the experimental groups consumed high-calorie choline-deficient diet (HCCDD), in which fat content was 45%, fructose content - 20% of the energy value of the diet. Rats of the 2nd group were fed HCCDD without any supplements, the 3rd group - with the addition of carnosine (75 mg/kg body weight), the 4th group - with the addition of α-lipoic acid (75 mg/kg body weight), the 5th group - with the combined addition of carnosine and α-lipoic acid in a total dose of 150 mg/kg body weight. Hematological values were determined on a hematological analyzer. The content of ghrelin and leptin, as well as growth factors GM-CSF and M-CSF in blood plasma and adipose tissue lysates, was determined by multiplex immunoassay using xMAP technology. Results. Rat intake of HCCDD resulted in decreased hemoglobin levels and red blood cell scores compared to controls. Diet enrichment with carnosine and α-lipoic acid did not have a reliable effect on these indicators. Carnosine intake had a protective effect on erythrocyte volume, a decrease of which was recorded in other experimental groups. HCCDD stimulated the growth of the absolute number of leukocytes in peripheral blood due to granulocytes and mononuclears. The enrichment of HCCDD with carnosine and α-lipoic acid led to a further increase in leukocytosis, the maximum level of which was observed in the group of rats fed HCCDD, simultaneously enriched with carnosine and α-lipoic acid (14.86±1.48×109/l compared to 8.67±1.23×109/l in control). All diets used in the research had no effect on the number of erythrocytes and platelets in the peripheral blood of rats. The use of both HCCDD alone and in combination with carnosine or α-lipoic acid intake had a negative effect on the level of growth factors GM-CSF and M-CSF in blood plasma and adipose tissue. The consumption of HCCDD caused an increase in leptin blood level (8.54±0.69 compared to 2.58±0.37 pg/ml in control, р<0.05), which was normalized by enriching the diet with carnosine and α-lipoic acid. Ghrelin blood level significantly decreased in all experimental groups compared to the control: by 30% in rats fed and by almost 50% when carnosine and α-lipoic acid were added to HCCDD. The intake of α-lipoic acid led to hormone level changes in adipose tissue lysates, leptin content decreased (2.31±0.11 vs 2.77±0.15 pg/ml), while ghrelin level significantly increased (0.35±0.14 vs 0.20±0.06 pg/ml), compared with the control group (р<0.05). Conclusion. The revealed interrelation of parameters of the cellular composition of peripheral blood and hemoglobin content with the changes in the content of GM-CSF, M-CSF, leptin and ghrelin in blood plasma and adipose tissue indicates the mutual influence of the studied CSF, leptin, ghrelin and added antioxidants (carnosine and α-lipoic acid) on the regulatory mechanisms of hematopoiesis in rats fed HCCDD.

Leukemia risk and rate of radiation dose accumulation. Part 2: Comparative analysis of leukocytic indices and dynamics of peripheral blood values in relation to external gamma-exposure dose

The results of a retrospective analysis of leukocytic indices and dynamics of peripheral blood values in relation to external gamma-exposure dose among the personnel of the first atomic production facility in Russia were presented. The study was performed on the basis of the database “Leukemia in the cohort of workers of the Mayak Production Association employed in 1948-1958”. The database contains hematological, clinical and dosimetry information on the two groups of workers: the study group includes individuals with leukemia as the cause of death (n=84); control group includes personnel without hematological cancer pathology (n=300). The control group was composed by selecting 3-4 internal control cases for each leukemia case taking into account gender, age of the start of the exposure and the same accumulated absorbed dose of gamma-exposure to red bone marrow. Based on 19592 analysis of peripheral blood we had described hematopoiesis shifts along cell lines, performed correlation analysis of interrelation between hemogram values and radiation dose, analyzed particular characteristics of blast cells’ appearance in peripheral blood flow in the period prior to leukemias. Comparative analysis of various leukocytic indices was performed among Mayak PA personnel for the first time. The diverse nature of the response of bone marrow hematopoiesis was noted; more pronounced hematological shifts were indicated regarding more intensive production exposure. A number of hematopoietic features were detected among people exposed to production radiation: 1) cytopenia in relation to minimum accumulated doses of radiation exposure in both studied groups; 2) more pronounced lability of hemograms in relation to increased accumulation dose of external gamma-exposure and period of radiation exposure among individuals who had developed leukemia in future in comparison to controls; 3) correlation between peripheral blood cells and accumulated doses of external gamma-exposure was characterized by the highest values of coefficients among individuals with future leukemias; 4) myeloblasts were most often registered among all the blast cells; proerythroblasts were characterized by the shortest average period from the start of the exposure and to the moment of their appearance in the peripheral blood flow (178 days in the study group); polychromatophile erythroblasts in peripheral blood were registered only among the workers with future leukemia; 5) when comparing leukocytic indices in proportion to the categories of accumulated doses of external gamma-exposure, a pronounced variability in the ratio of blood cells was observed among exposed individuals who were subsequently diagnosed with leukemia, especially in the range of 2.0-2.5 Gy. Thus, a comprehensive analysis of hematological parameters, including the assessment of leukocytic indices, is more informative than studying a standard hemogram. Particular features of the dynamics of the cellular composition of peripheral blood can be used as indicators of pathological hematopoiesis in exposed workers prior to clinical manifestation of leukemia.

Analysis of systemic immunity and inflammation in the prognosis of gastric adenocarcinoma

Objective: to study the state of cellular immunity in patients with gastric adenocarcinoma.Materials and methods. From 2017 to 2018, 45 previously untreated patients with gastric adenocarcinoma (25 with stage I–III, 20 with stage IV) received surgical / combined treatment or independent chemotherapy, respectively, at the N. N. Blokhin National Medical ResearchCenter of Oncology. Peripheral blood sampling was carried out before starting treatment. We studied the cellular composition of peripheral blood, as well as systemic immunity parameters determined by flow cytometry (CD3+CD4+; CD3+CD8+; CD4+CD8+; CD4+/CD8+; CD3–CD16+CD56+; CD3–CD19+), and their prognostic significance in relation to overall survival and progression-free survival.Results. The prognostic value of the relative indicator of platelet lymphocytic index was determined: progression-free survival in patients with a high level of platelet-lymphocytic index (&gt;208.7) was higher: 8.1 months versus 4.5 months (p = 0.0027). A favorable prognosis for overall survival was an increase in the number of CD3–CD19+ lymphocytes (hazard ratio (HR) 0.91; 95 % confidence interval (CI) 0.85–0.97; p = 0.007), and an unfavorable prognosis was an increase in the number of neutrophils (HR 1.26; 95 % CI 1.05–1.50; p = 0.012), platelet count (HR 1.01; 95 % CI 1.0–1.01; p = 0.043), as well as an increase in the number of NK cells (HR 1.04, 95 % CI 1.0–1.09; p = 0.029).Conclusion. Indicators of the cellular composition of peripheral blood, characterizing a systemic inflammatory reaction, as well as indicators of systemic immunity, can serve as additional prognostic factors for gastric cancer.

Experimental evaluation of the effectiveness of a damage control surgery in combined radiation-mechanical injuries

Relevance. Currently, damage control surgery (DCS) is actively used in polytrauma. However, data on the effectiveness of this tactic for combined radiation-mechanical injuries (CRMI) in the available literature was not found.Objective. To develop a model of CRMI on small laboratory animals (rabbits), which allows studying the effectiveness of the use of DCS in comparison with the early total care surgery tactics.Methods. A model of a severe CRMI was proposed. The animals were subjected to a generally uniform short-term gammairradiation, and then they suffered a liver injury on the background of blood loss of 40% of the blood volume and hypothermia. After modeling the lesion, a comparison was made of the effectiveness of the tactics of the DCS and the early total care surgery tactics. During the study, the following indicators were evaluated: the average life expectancy and mortality of animals, changes in the cellular composition of peripheral blood, features of behavioral reactions to irradiation, the amount of blood loss during the implementation of the surgical intervention.Results and their analysis. The experiment showed that the tactics of the DCS have an advantage over the traditional tactics of a one-time exhaustive operation in the case of the CRMI. When performing DCS, there was no death of rabbits on the 1st day after the operation, when implementing early total care surgery - 3 rabbits died, besides, the mortality was absolute, while with DCS one rabbit survived (12.5%). The final operation at the DCS occurs prior to the beginning of the acute period of CRMI, which corresponds to the requirements of the instructions for the treatment of CRMI. In the course of the study, it was found that the proposed model of CRMI contributes to the formation of the phenomenon of mutual burdening and, as a result, complicates both the radiation and mechanical components of the lesion occurs.Conclusion The proposed experimental model for the application of severe CRMI helps to explore DCS tactics in the treatment of this pathology. DCS is a promising method of surgical treatment of CRMI, however, for more accurate extrapolation of data from animal to human, additional research on large laboratory animals is required.

The alterations of cellular composition of blood and hormonal balance under adenocarcinoma of ascending part of colon as diagnostic criteria of development of processes of metastasis.

The article presents the analysis of character of derangement of peripheral blood composition and blood immune status and their relationship with hormonal imbalance. The complex clinical laboratory examination was carried out covering 60 patients with local (T1-2 N0M0) and locally prevalent (T3-4 (а, b) N0M0, T3-4 (а, b) N1-2M0) stages of adenocarcinoma of ascending section of colon residing for treatment in the clinic of surgery and oncology at the base of Non-State Institution of Health Care "The Road Clinical Hospital at the Station Saratov II" of The Public Corporation "Russian Railways" during 2012-2016. The diagnosis was verified using such common technique as colonoscopy with express-cytological analysis. The distribution of patients on observation groups was implemented after operative treatment (right-side hemicolectomy) and by results of planned pathomorphological analysis. The comparative evaluation was carried out concerning cellular composition of peripheral blood, sub-population composition of lymphocytes and content in blood of adrenocorticotropin, cortisol and somatotropic hormone. The pathogenic relationship is established between progressively increasing of level of cortisol in blood of patients, immunodeficiency on T- and then on B-system of lymphocytes, increasing of level of somatotropic hormone and development of local metastases. The diagnostic criteria of transition of local stage of adenocarcinoma of ascending part of colon to locally prevalent form is progressively increasing content of cortisol, adrenocorticotropin and somatotropic hormone in blood against the background of increasing lymphopenia, monocytopenia, T-dependent immunodeficiency. The monitoring of indices of level of lymphocytes, monocytes of blood and their sub-population composition, content of cortisol and somatotropic hormone in blood can be used as criteria of prevalence of neoplasia in case of clinically non-manifesting metastases.

Rapid changes in peripheral lymphocyte concentrations during interferon-free treatment of chronic hepatitis C virus infection.

Treatment of chronic hepatitis C virus (HCV) infection with direct‐acting antivirals results in a rapid decline in viral load and markers of hepatic inflammation, including serum chemokine (C‐X‐C motif) ligand 10 (CXCL10) concentration, which is followed in most cases by a sustained virologic response. Whether parallel changes of significance occur in the cellular composition of peripheral blood is relatively unknown. We hypothesized that longitudinal characterization of peripheral blood during treatment would provide insight into cellular migration and immune activation, which would have implications for understanding host immunity both before and after HCV treatment and may relate to HCV clearance. We analyzed longitudinal peripheral innate and adaptive immune cell populations by flow cytometry from 95 subjects enrolled in two direct‐acting antiviral clinical trials and examined chemokine receptor expression on T lymphocytes in 43 patients. Within 1‐2 weeks of initiating treatment, significant increases were observed in the concentration of peripheral cluster of differentiation 4–positive (CD4+) and CD8+ T lymphocytes but not monocyte or natural killer cells. In tandem with these changes, the percent of both CD4+ and CD8+ T lymphocytes with an activated phenotype (human leukocyte antigen [HLA] DR+ and CD38+) decreased, and T‐lymphocyte surface expression of chemokine (C‐X‐C motif) receptor 3, the chemokine receptor for CXCL10, increased. Conclusion: Rapid changes in peripheral cellular populations occur during direct‐acting antiviral treatment of HCV infection, which could potentially relate to hepatic efflux of tissue lymphocytes due to altered inflammation and chemokine receptor signaling, providing critical insight into the relationship between host immunity and viral clearance during HCV infection. (Hepatology Communications 2017;1:586–594)

Impaired bone healing in multitrauma patients is associated with altered leukocyte kinetics after major trauma.

Animal studies have shown that the systemic inflammatory response to major injury impairs bone regeneration. It remains unclear whether the systemic immune response contributes to impairment of fracture healing in multitrauma patients. It is well known that systemic inflammatory changes after major trauma affect leukocyte kinetics. We therefore retrospectively compared the cellular composition of peripheral blood during the first 2 weeks after injury between multitrauma patients with normal (n=48) and impaired (n=32) fracture healing of the tibia. The peripheral blood-count curves of leukocytes, neutrophils, monocytes, and thrombocytes differed significantly between patients with normal and impaired fracture healing during the first 2 weeks after trauma (P-values were 0.0122, 0.0083, 0.0204, and <0.0001, respectively). Mean myeloid cell counts were above reference values during the second week after injury. Our data indicate that leukocyte kinetics differ significantly between patients with normal and impaired fracture healing during the first 2 weeks after major injury. This finding suggests that the systemic immune response to major trauma can disturb tissue regeneration.

The influence of electrical stimulation on the peripheral immune system under the experimental and clinical conditions

The authors for the first time provide the scientifically-grounded substantiation for the application of electrical stimulation with bipolar pulsed currents for the combined treatment of the patents presenting with acne vulgaris. Experiments on the rabbits have demonstrated the influence of bipolar pulsed currents on the cellular composition of lymph nodes and thereby facilitated the better understanding of certain theoretical aspects of the application of electrical stimulation at large. The clinical study on the application of multi-channel electrical stimulation and microcurrent therapy showed that these physical factors may cause remodeling of immunogenesis in the patents presenting with acne vulgaris manifested as the normalization of the cellular composition of peripheral blood (leukocyte, lymphocyte, T- and B-lymphocyte counts and immunoglobulin levels). These findings confirm the effectiveness of the proposed integrated treatment responsible for the enhancement of the overall resistance of the patents with acne vulgaris. Moreover, this therapeutic modality proved to exert the immunocorrective action and promote the restoration of the adaptive capacity at large. As a result, 80.9% of the patients presenting with acne vulgaris enjoyed prolonged remission of the disease.

Age-specific features in the adaptation response of peripheral blood cells to a recurrence of chronic obstructive pyelonephritis in women

The goal of this research was to evaluate adaptative changes in the cellular composition of peripheral blood in a recurrence of inflammation in elderly women with chronic obstructive pyelonephritis (COP). We analyzed clinical laboratory data on 50 women with COP, distributing the subjects according to two age periods: a control group comprising women younger than 55 (47 ± 3.22 years; n = 22), and a main group formed of women aged over 65 (70.5 ± 1.6 years; n = 28). During a recurrence of COP, the women over 65 had higher counts of leukocytes, neutrophils, monocytes, and the erythrocyte sedimentation rate (ESR), compared to their remission levels, by 90.1, 20.8, 71.4, and 31.6%, respectively, whereas the count of lymphocytes was 2.8 times lower. A recurrence of COP in women under 55 caused a 87% growth in the count of leukocytes (p 0.05) against the remission values. The higher expression of neutrophilosis and lymphocytopenia manifestations can also be attributed to age-specific features in the blood cell adaptation response to a recurrence of COP.

Peromyscus leucopus mice: a potential animal model for haematological studies.

Peromyscus leucopus mice share physical similarities with laboratory mice Mus musculus (MM) but have higher agility and longer lifespan. We compared domesticated P. leucopus linville (PLL) and M. musculus C57BL/6 (MMB6) mice for cellular composition of peripheral blood (PB), bone marrow (BM) and spleen. PLL mice had significantly fewer platelets and significantly more monocytes in the blood, and notably fewer megakaryocytes in the BM. Spleens of PLL mice were significantly smaller, with 50% fewer cells and reduced 'red pulp'. There was no obvious haematological change in PLL mice between 2-8 and 16-26 months of age, except for a significant increase in blood monocytes. Cellular reactive oxygen species (ROS) content showed no change with age but differed significantly between different cell types. Treating two to eight month-old PLL mice with antioxidant N-acetylcysteine in drinking water for three months did not affect cellular ROS content, but increased blood leucocytes especially the concentration of monocytes. The low platelets, low megakaryocytes, high monocytes and low splenic erythropoiesis in PLL mice resemble human measurements better than the values seen in MMB6.

Femoral bone marrow aspiration in live mice.

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

Кислотно-гемолитическая устойчивость эритроцитов напряженного эритропоэза в условиях низкочастотной вибрации

Исследовано 14-дневное влияние вибрации с частотами 8, 16, 24 и 32 Гц, амплитудой 0,8±0,12 мм на кислотную резистентность эритроцитов мышей. Вибрация с частотами 8, 16 и 24 Гц вызывает напряженный эритропоэз. Высокая активность стресс-реализующих механизмов приводит к падению общего содержания эритроцитов в крови, снижению доли «старых», низкостойких эритроцитов и общему повышению их устойчивости к кислотному гемолизу. Вибрация с частотой 32 Гц блокирует эритропоэз, следствием чего является быстрое истощение резерва зрелых эритроцитов и снижение их кислотной устойчивости.