T-cell Content Research Articles

Clinical and immunological characteristics of moderate-to-severe forms of COVID-19 at different levels of the tissue damage marker (lactate dehydrogenase)

Objective: To study the features of the immune status in patients with a moderate-to-severe course of COVID-19, depending the levels of blood lactate dehydrogenase.Materials and Methods: A total of 24 patients with a moderate-to-severe form of COVID-19 were examined. The control group consisted of 21 healthy volunteers. Methods: clinical, paraclinical (computed tomography of the lungs; complete blood count, blood biochemistry; immunological studies), statistical.Results: Changes in complete blood count and blood biochemistry in patients with moderate-to-severe COVID-19 consist in granulocytosis, lymphopenia, monocytopenia, and an increase in the level of C-reactive protein, lactate dehydrogenase, with a simultaneous decrease in the total protein content. In patients with high levels of lactate dehydrogenase, a redistribution of lymphocyte populations towards B-cells was revealed with a decrease in the total number of T-cells. At the same time, there was a decrease in the production of IgM and IgG and a simultaneous increase in the synthesis of IgA.Conclusions: The increase in blood LDH in COVID-19 patients is associated with a decrease in the content of T-cells due to severe lymphopenia, and a simultaneous increase in the content of B-cells without adequate enhancement of their antibody production function.

Peculiarities of the immune status of patients with multidrug­resistant pulmonary tuberculosis after application of treatment regimens with bedaquiline and linezolid

Objective — to investigate immuno-metabolic homeostasis in patients with new and recurrent cases of destructive forms of multidrug-resistant pulmonary tuberculosis (MDR-PTB) after treatment with bedaquiline and linezolid. Materials and methods. A clinical and laboratory examination of 175 people with MDR-PTB (89 patients with new cases and 86 patients with recurrent cases of destructive forms of MDR-PTB). The study was performed before and after treatment with bedaquiline and linezolid. The total leukocytes count (L), the leukocyte differential count (leukocyte formula), was determined in all subjects, The content of populations and subpopulations of lymphocytes were calculated using monoclonal antibodies to CD3+, CD4+, CD8+, CD19+, CD23+, CD56+ lymphocytes antigen in the reaction of indirect immunofluorescence. Quantitative determination of serum immunoglobulins was performed by Manchini radial immunodif­fusion in a gel. The level of circulating immune complexes (CIC) was determined by the spectrophoto­metric method by precipitation in polyethylene glycol. Results and discussion. In patients with new cases of destructive forms of MDR-PTB to treatment revealed hypersensitivity reactions of the first and the fourth type. Expressed activation of humoral and killer parts of immunity detected. In patients with recurrent cases of destructive forms of MDR-PTB before treatment, there are pronounced hypersensitivity reactions of the fourth type and activation of the humoral and killer parts of the immune system.Patients with new cases of destructive forms of MDR-PTB after application of treatment regimens with bedaquiline and linezolid have hypersensitivity reactions of the first and fourth types. Activation of humo­ral and killer parts of immunity detected. In patients with recurrent cases of destructive forms of MDR-PTB after the application of treatment regimens with bedaquiline and linezolid revealed a pronounced hypersensitivity reaction of the first type and activation of the killer and humoral parts of the immune system. Conclusions. In patients with new cases of destructive forms of MDR-PTB before treatment revealed T-cell immunodeficiency, which is expressed by a decrease in the level of T-lymphocytes (1.7 times relative to normal) and T-helpers (twice below normal). In patients with recurrent cases of destructive forms of MDR-PTB before treatment revealed T-cell immunodeficiency with a marked decrease in the level of T-lymphocytes (1.8 times normal) and T-helpers (1.8 times below normal). In patients with new cases of destructive forms of MDR-PTB after the application of treatment regimens with bedaquiline and linezolid revealed activation of the T-cell immune system due to increased levels of T-suppressors. In patients with recurrent cases of destructive forms of MDR-PTB after the application of treatment regimens with bedaquiline and linezolid revealed T-cell immunodeficiency due to the reduced content of T-lymphocytes (1.5 times below normal), namely T-helpers (1.9 times lower than normal).

Characterization of m6A-Related Genes Landscape in Skin Cutaneous Melanoma to Aid Immunotherapy and Assess Prognosis.

BackgroundSkin cutaneous melanoma (SKCM) is the most malignant tumor among skin cancers. Immunotherapy has shown a great role in the advantageous prognosis of SKCM. However, only a small percentage of people can benefit from immunotherapy. To date, there has been insufficient evidence to reveal the prognostic value of m6A in SKCM and its relationship with the infiltration of immune cells and the efficacy of immunotherapy.MethodsHere, we synthetically analyzed 23 m6A regulators from SKCM samples collected from the TCGA and GEO databases. We defined three m6A modification patterns and constructed m6A scores using principal component analysis (PCA).ResultsWe found significant differences in overall survival (OS) and immune infiltration between different m6A subclusters. Besides, m6A score was positively correlated with regulatory T-cell and helper T-cell content, which may account for the association of high m6A scores with superior prognosis. Multivariate Cox regression analysis revealed that the m6A score was an independent prognostic indicator. Moreover, patients with high m6A scores showed a better response to immunotherapy, and this result was further validated in two independent immunotherapy cohorts receiving anti-PD-1/PD-L1 therapy.ConclusionThe findings suggested the m6A score can screen suitable candidates for immunotherapy and can predict immunotherapy response. This analysis of different m6A patterns in a large sample of SKCM expanded our understanding of TME and provided new ideas for prognostic assessment and personalized immunotherapy strategies for SKCM patients.

Immunity indicators in tuberculosis patients with diabetes mellitus and autoimmune thyroiditis

Objective — to study the parameters of immunity in tuberculosis with diabetes mellitus and with subclinical hypothyroidism against the background of autoimmune thyroiditis. 
 Materials and methods. 60 patients with pulmonary tuberculosis and diabetes mellitus were under observation. In 30 patients with destructive tuberculosis and diabetes mellitus against the background of autoimmune thyroiditis with subclinical hypothyroidism, the parameters of humoral and cellular immunity were studied. The control group consisted of 30 patients with destructive tuberculosis and diabetes mellitus with intact structure and function of the thyroid gland. In patients of both groups, we assessed the state of T- and B-systems of immunity, natural killers. 
 Results and discussion. The phenotyping of lymphocytes was carried out and the relative content of T-cells was determined: CD3+, T-helpers (CD4+), cytotoxic T-cells, B-lymphocytes (CD19+) and natural killer cells (CD16+). Using the enzyme immunoassay, the cytokine profile was determined: the levels of tumor necrosis factor-α, interferon-γ, interleukin-2, -6 and -4. It has been established that in patients with diabetes mellitus, both cellular and humoral immunity are suppressed. But a significantly more significant suppression of immunity was diagnosed in patients with tuberculosis against the background of diabetes mellitus and autoimmune thyroiditis with subclinical hypothyroidism. 
 Conclusions. According to the authors, it is the deficiency of thyroid hormones that is the negative effect of a more significant suppression of immunity in patients autoimmune thyroiditis with subclinical hypothyroidism.

A Subset of Localized Prostate Cancer Displays an Immunogenic Phenotype Associated with Losses of Key Tumor Suppressor Genes.

A subset of primary prostate cancer expresses programmed death-ligand 1 (PD-L1), but whether they have a unique tumor immune microenvironment or genomic features is unclear. We selected PD-L1-positive high-grade and/or high-risk primary prostate cancer, characterized tumor-infiltrating lymphocytes with multiplex immunofluorescence, and identified genomic alterations in immunogenic and nonimmunogenic tumor foci. One quarter of aggressive localized prostate cancer cases (29/115) had tumor PD-L1 expression more than 5%. This correlated with increased density of CD8+ T cells, a large fraction coexpressing PD-1, versus absent PD-1 expression on sparse CD8 T cells in unselected cases. Most CD8+PD-1+ cells did not express terminal exhaustion markers (TIM3 or LAG3), while a subset expressed TCF1. Consistent with these CD8+PD-1+TCF1+ cells being progenitors, they were found in antigen-presenting cell niches in close proximity to MHC-II+ cells. CD8 T-cell density in immunogenic prostate cancer and renal cell carcinoma (RCC) was nearly identical. Shallow RB1 and BRCA2 losses, and deep deletions of CHD1, were prevalent, the latter being strongly associated with a dendritic cell gene set in The Cancer Genome Atlas. Tumor mutation burden was variable; neither high microsatellite instability nor CDK12 alterations were present. A subset of localized prostate cancer is immunogenic, manifested by PD-L1 expression and CD8+ T-cell content comparable with RCC. The CD8+ T cells include effector cells and exhausted progenitor cells, which may be expanded by immune checkpoint inhibitors (ICI). Genomic losses of RB1, BRCA2, and CHD1 may be drivers of this phenotype. These findings indicate that immunotherapies may be effective in biomarker-selected subpopulations of patients with localized prostate cancer.

A tyrosine kinase inhibitor-induced interferon response positively associates with clinical response in EGFR-mutant lung cancer

Tyrosine kinase inhibitors (TKIs) targeting EGFR-mutant lung cancers promote a range of tumor regression responses to yield variable residual disease, a likely incubator for acquired resistance. Herein, rapid transcriptional responses induced by TKIs early in treatment that associate with the range of patient responses were explored. RNAseq was performed on EGFR mutant cell lines treated in vitro with osimertinib and on tumor biopsies of eight EGFR mutant lung cancer patients before and after 2 weeks of TKI treatment. Data were evaluated for gene expression programs altered upon TKI treatment. Chemokine and cytokine expression were measured by ELISA and quantitative RT-PCR. IκB Kinase (IKK) and JAK-STAT pathway dependence was tested with pharmacologic and molecular inhibitors. Tumor sections were stained for the T-cell marker CD3. Osimertinib stimulated dynamic, yet wide-ranging interferon (IFN) program regulation in EGFR mutant cell lines. IL6 and CXCL10 induction varied markedly among the EGFR mutant cell lines and was sensitive to IKK and JAK-STAT inhibitors. Analysis of matched patient biopsy pairs revealed marked, yet varied enrichment of IFN transcriptional programs, effector immune cell signatures and T-cell content in treated tumors that positively correlated with time to progression in the patients. EGFR-specific TKIs induce wide-ranging IFN response program activation originating within the cancer cell. The strong association of IFN program induction and duration of clinical response indicates that the TKI-induced IFN program instructs variable recruitment and participation of immune cells in the overall therapeutic response.

Associations Among Adipose Tissue Immunology, Inflammation, Exosomes and Insulin Sensitivity in People With Obesity and Nonalcoholic Fatty Liver Disease

Insulin resistance is a key factor in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). We evaluated the importance of subcutaneous abdominal adipose tissue (SAAT) inflammation and both plasma and SAAT-derived exosomes in regulating insulin sensitivity in people with obesity and NAFLD. Adipose tissue inflammation (macrophage and T-cell content and expression of proinflammatory cytokines), liver and whole-body insulin sensitivity (assessed using a hyperinsulinemic-euglycemic clamp and glucose tracer infusion), and 24-hour serial plasma cytokine concentrations were evaluated in 3 groups stratified by adiposity and intrahepatic triglyceride (IHTG) content: (1) lean with normal IHTG content (LEAN; N= 14); (2) obese with normal IHTG content (OB-NL; N= 28); and (3) obese with NAFLD (OB-NAFLD; N= 28). The effect of plasma and SAAT-derived exosomes on insulin-stimulated Akt phosphorylation in human skeletal muscle myotubes and mouse primary hepatocytes was assessed in a subset of participants. Proinflammatory macrophages, proinflammatory CD4 and CD8 T-cell populations, and gene expression of several cytokines in SAAT were greater in the OB-NAFLD than the OB-NL and LEAN groups. However, with the exception of PAI-1, which was greater in the OB-NAFLD than the LEAN and OB-NL groups, 24-hour plasma cytokine concentration areas-under-the-curve were not different between groups. The percentage of proinflammatory macrophages and plasma PAI-1 concentration areas-under-the-curve were inversely correlated with both hepatic and whole-body insulin sensitivity. Compared with exosomes from OB-NL participants, plasma and SAAT-derived exosomes from the OB-NAFLD group decreased insulin signaling in myotubes and hepatocytes. Systemic insulin resistance in people with obesity and NAFLD is associated with increased plasma PAI-1 concentrations and both plasma and SAAT-derived exosomes. ClinicalTrials.gov number: NCT02706262 (https://clinicaltrials.gov/ct2/show/NCT02706262).

Прогнозування тяжких нападів астми фізичного навантаження у дітей за імунологічними показниками крові

Bronchial asthma is considered an umbrella diagnosis for various diseases with certain pathophysiological mechanisms (endotypes) and variable clinical manifestations (phenotypes). The phenotype of exercise-induced asthma is one of the most common variants of the disease in children and adolescents. The treatment tactic of acute asthma attack is unified and depends of the severity in contrast to preventive therapy the vector of which provides an individualized approach. However, the issues concerning the duration, frequency of administration and amount of drugs prescription not finally resolved. The purpose of the study was to evaluate the blood immunological parameters in patients with exercise-induced asthma phenotype to predict the severity of disease exacerbation and optimize the reliever therapy. Material and methods. We examined 43 children suffering from exercise-induced asthma. Two clinical groups were formed according to the severity of the exacerbation. The first group included 22 patients with moderate exacerbation of asthma, and the second group had 21 children with severe symptoms of the disease, which was interpreted as a severe asthma attack. There were not any significant differences by sex, place of residence, age and diseases duration among comparison groups. The obtained results showed a significant increase in the level of B-lymphocytes in patients of first clinical group (26.64 %) compared with children with severe exacerbation (20.40 %), increase content of T-lymphocytes (due to T-helper cells) in schoolchildren of the second clinical group (42.32 %) compared with patients with moderate asthma attack (33.32 %). The attributive risk of more severe disease exacerbation with B-lymphocytes ≤20 % and T- helper cells ≥20 % was 36.6 %, the relative risk was 1.9 with odds ratio 5.0. A significantly higher level of pharmazan-positive neutrophils in spontaneous and stimulated modifications of the nitro blue tetrazolium test was recorded in patients with moderate exacerbation (35.18 % and 45.09 %) compared with children of the second clinical group (19.33 % and 27.81 %). The content of neutrophils in the spontaneous nitro blue tetrazolium test ≤15 % allowed evaluating severe compared with moderate asthma attack with specificity of 95.5 %, positive predictive value of 90.9 %, and positive likelihood ratio 10.5. Conclusion. The use of certain immunological parameters allows working out an individual plan for asthma attacks and may reduce the frequency of severe exacerbations in children suffering from exercise-induced bronchial asthma

Characterization of T cell, B cell and Natural Killer Cell Subsets in COVID-19 Patients

Immune dysregulation is observed in patients with severe COVID-19 associated pneumonia. The aim of the investigation was to perform analysis of immune parameters including cell count, differentiation and activation of lymphocytes from COVID-19 patients. 57 patients with PCR and chest CT confirmed diagnosis of COVID-19 pneumonia were included in the study. Patients were divided into 2 groups: severe/critical (S/C) patients (n=18) which were treated in ICU and required IMV; and moderate (M) patients (n=39). Blood samples from 19 healthy donors (HD) controls were obtained. T cell, B cell, natural killer (NK) cell and innate lymphoid cell (ILC) subsets were assayed using Attune NxT flow cytometer. Nonparametric statistics were used. A profound decrease of the absolute counts of T-cells, B-cells, NK- and NKT-cells and frequencies (%) of ILC was noted both in S/C and M versus HD (p<0.01). Frequency (%) of B-cells in S/C was increased. A decrease in the content of naive T-cells was noted, while the content of TCM, TEM, TEMRA, T-reg and activated HLA-DR+ T-cells did not change significantly in S/C compared with M but both groups had increased exhausted PD-1+ T cells counts. T and NK cell lymphopenia was observed in COVID-19 patients, more significant in S/C. No important changes in T-cells differentiation and activation were detected in with S/C or M groups accompanied by increase in exhausted T cells. Immune system anergy may explain the severe course of some COVID 19 patients and cases of reinfection.

Immune cell analysis of pilocytic astrocytomas reveals sexually dimorphic brain region-specific differences in T-cell content.

Immune cell analysis of pilocytic astrocytomas reveals sexually dimorphic brain region-specific differences in T-cell content.

Immunopathogenetic features and prognostic criteria for severe hemorrhagic fever with renal syndrome

Aim. Assessment of the features of cellular immunological mechanisms at the early stage of HFRS of varying severity and development of prognostic criteria for the risk of a severe course of the infectious process. Materials and methods. An immunological blood test (flow cytofluorimetry method) was performed in 12 patients with severe HFRS and 53 patients with moderate course in the dynamics of the disease. Statistical data processing was performed based on the SPSS software package. Results. At the initial stages of HFRS, immunological features of the severe course of the disease were established in the form of a higher content of T-helper and regulatory T-cells in the blood and a reduced number of CTL, including their activated pool. Based on these changes, an immunological prognostic coefficient of HFRS was developed, which allows determining the risk of severe course in the early days (febrile period) of the disease with high prognostic accuracy. Conclusion. The results obtained allowed us to identify previously unknown features of the immune process at the initial stages of HFRS development, which allowed us to propose a new approach to predicting the severe course of the disease.

Clinical Impact of Tumor-Associated Macrophage and T-Cell Contents in Diffuse Large B-Cell Lymphoma

Background: Tumor infiltrating immune cells can modulate cancer progression and are attractive therapeutic targets. We have previously identified a tumor microenvironment (TME) immune cell signature in diffuse large B-cell lymphoma (DLBCL), which contains genes for cytolytic factors, immune checkpoint molecules, T-cells and macrophages, and separates the patients into immune cell high and low or T-cell inflamed and non-inflamed subgroups (Autio et al., 2020). As macrophages and T-cells are key components of the TME, our aim was to characterize their phenotypes and relationships in the tumor tissue and associate the findings to clinical outcome in patients with DLBCL. Methods: We used multiplex immunohistochemistry, gene expression data, and unsupervised computational approaches to characterize tumor associated T-cell and macrophage (TAM) phenotypes in 178 samples from DLBCL patients. We correlated the immune cell constitution with clinical data, and validated the findings utilizing gene expression data from three independent DLBCL cohorts (Reddy et al., 2017, Schmitz et al., 2018, Chapuy et al., 2018) and in silico immunophenotyping with CIBERSORT. Results: The proportions of TAMs and infiltrating T-cells varied significantly between the patients. Unsupervised hierarchical clustering divided the samples into T-cell inflamed and non-inflamed subgroups. The T-cell inflamed phenotype was rich in other immune cell subtypes such as TAMs, natural killer (NK) cells, and regulatory T-cells, thus characterizing an immune hot phenotype. Apart from B-symptoms being more common in the patients with T-cell inflamed phenotype, clinical characteristics were equally distributed between the subgroups, and no association with survival was observed. However, when we divided the T-cell inflamed group further into subgroups according to the TAM content, we identified a T-cell high/macrophage low group, which was characterized by clinically less aggressive disease and better survival as compared to the T-cell high/macrophage high or T-cell low groups. Using in silico deconvolution analyses, we validated the T-cell high/macrophage low subgroup, and its association with less aggressive disease and survival in an external dataset comprising 496 patients (Reddy et al., 2017). The prognostic impact of T-cell high/macrophage low subgroup on survival was independent of the IPI and molecular subtype. Differential gene expression analyses identified a gene signature corresponding to the T-cell high/macrophage low subgroup. This signature translated to better outcome, and was validated in two additional datasets (Schmitz et al. n=562, Chapuy et al. n=137). Finally, we studied the impact of immune checkpoint expressing TAMs on survival, and whether it differed between the T-cell inflamed and T-cell non-inflamed subgroups. In particular, a high proportion of PD-L1+, TIM3+, and PD-L1+TIM3+CD163- macrophages associated with poor outcome, independent from the IPI and molecular subtype. Furthermore, the impact of these TAM subtypes on survival was evident only in the patients with T-cell inflamed phenotype. Conclusions: Our data demonstrate that the proportions of different immune cell phenotypes in the DLBCL TME are clinically meaningful, and suggest that the high T-cell/macrophage ratio predicts favorable survival in patients with DLBCL. Disclosures Leppa: Novartis: Consultancy; Takeda: Consultancy; Celgene: Research Funding; Bayer: Research Funding; Incyte: Consultancy; Roche: Consultancy, Research Funding.

Melanoma brain metastases have lower T-cell content and microvessel density compared to matched extracranial metastases

BackgroundAlthough melanoma brain metastases (MBM) tend to respond to systemic therapy concordantly with extracranial metastases, little is known about differences in immune cell and vascular content between the brain and other metastatic sites. Here we studied infiltrating immune cell subsets and microvessel density (MVD) in paired intracerebral and extracerebral melanoma metastases.MethodsPaired intracerebral and extracerebral tumor tissue was obtained from 37 patients with metastatic melanoma who underwent craniotomy between 1997 and 2014. A tissue microarray was constructed to quantify subsets of tumor-infiltrating T-cell, B-cell, and macrophage content, PD-L1 expression, and MVD using quantitative immunofluorescence.ResultsMBM had lower CD3+ (p = 0.01) and CD4+ (p = 0.003) T-cell content, lower MVD (p = 0.006), and a trend for lower CD8+ (p = 0.17) T-cell content compared to matched extracerebral metastases. There were no significant differences in CD20+ B-cell or CD68+ macrophage content, or tumor or stroma PD-L1 expression. Low MVD (p = 0.008) and high CD68+ macrophage density (p = 0.04) in intracerebral metastases were associated with improved 1-year survival from time of first MBM diagnosis.ConclusionsAlthough responses to immune-modulating drugs in the body and the brain tend to be concordant, differences were found in MVD and T-cell content between these sites. Studies of these markers should be incorporated into prospective therapeutic clinical trials to determine their prognostic and predictive value.

Expansion and Activation of Human Natural Killer Cells ex vivo in the Presence of Transgenic Feeder Cells

Preliminary results of clinical trials show that the use of activated and expanded ex vivo natural killer cells is a promising tool for adoptive immunotherapy of acute myeloid leukemia, as well as a number of solid tumors, when used together with targeted monoclonal antibodies. However, the introduction of this approach into clinical practice is limited by the possibility of obtaining a sufficient amount of the NK cell product with required characteristics. To solve this problem, we obtained transgenic feeder cells based on immortalized K562 cells expressing the recombinant membrane-bound variant of human interleukin-21 and 4-1BBL protein. Cocultivation of mononuclear cells obtained from the peripheral blood of ten healthy donors with genetically modified feeder cells resulted in significant expansion of natural killer cells (median expansion fold 21 589 times; minimum, 3150 times; maximum, 304 328 times) with a minimum content of T-cells (median, 0.5%; minimum, 0.06%; maximum, 4.7%) compared with their initial number. NK cells obtained in this way were not contaminated with the BCR–ABL1 oncogene from feeder cells and had a lower expression of c-MYC protooncogene compared to the initial level.

Hygienic assessment of water quality and its impact on the body of pigs

The results of research on the overall impact of drinking water quality, microclimate parameters and adopted in the farms of FE PE “Hlyniany Agro” and LLC “Miasni Resursy” options for growing weaned young (single- and three-phase) on morphological and biochemical parameters of blood, the level of natural resistance and their growth indicates that single-phase cultivation is more favorable for the growth and development of young animals. This is confirmed by the live weight of animals at birth and at the age of 120 days. Thus, in the case of single-phase cultivation of FE PE “Glynyany Agro” the live weight of piglets at birth was 1.11 kg, and in the case of three-phase LLC “Miasni Resursy” – 1.07 kg. When the animals reached 120 days of age, the live weight of piglets was 53.2 kg in FE PE “Glynyany Agro” and 50.2 kg in LLC “Miasni Resursy”. It should be noted that according to most indicators, water quality in both farms is within acceptable limits of sanitary and hygienic requirements, although it needs to be improved. With regard to microclimatic indicators, an overestimated level of ammonia was found in FE PE “Glynyany Agro”, which exceeded the MPC by 6 and 3.33 mg/m3, respectively, in the lines and transition periods, and in LLC “Miasni Resursy” was close to its upper borders. In a farm with single-phase animal husbandry, a slight decrease in erythrocytes in the blood of piglets aged 30 and 60 days was found. Since the animals reached the 120th day of life, an increase in this indicator was noted and the number of erythrocytes in the blood of animals of both farms, within the physiological norm, was 6.47 T/l (FE PE “Hlyniany Agro”) and 7.31 T/l (Miasni Resursy LLC). The concentration of hemoglobin was low in animals of both farms, but the highest rates were in animals of FE PE “Hlyniany Agro” at 30 and 60 days of age, and at 120 days of age this figure (3.28 g/l) was higher already in piglets of Miasni Resursy LLC. The content of total protein in the blood of piglets from both farms was also low, but by the time the animals reached 120 days of age, it was already within the physiological norm. Violation of the albumin-globulin ratio with the vast majority of globulins was also noted. Regarding cellular immunity, it was noted that the content of T-lymphocytes only in animals 120 days of age was normal, exceeding the lower limit of physiological norm by 0.27 % in animals with FE PE “Hlyniany Agro” and 2.09 % in animals with Miasni Resursy LLC. Our research once again confirms that the health, growth, development, and productivity of animals are influenced by a number of factors, both hygienic and technological.

IMMUNOLOGICAL PARAMETERS OF BONE MARROW IN NON-SMALL CELL LUNG CANCER

Introduction Generation of most immunocompetent cells takes place in bone marrow Bone marrow. As well, bone marrow is a peripheral lymphoid organ where antitumor effector cells and memory cells are present. The aim of the work is to estimate peripheral lymphoid cell subpopulations in bone marrow of lung cancer patients. Materials and methods Study has been done in 68 pts with lung cancer: squamous cell cancer (n = 28), adenocarcinoma (n = 38), other forms (n = 2). In all cases standard diagnostic and staging procedures were performed, as well as morphological (myelogram) and immunological study of bone marrow lymphocyte subpopulations. Multicolor Flow cytomtry was used for study of bone marrow lymphocyte populations. We studied T-cells and its subpopulations, B-cells, NK-cells, perforin-positive cells, and CD27-positive cells. Results Squamous cell lung cancer was characterized by higher content of bone marrow mature T-cells (CD3), and CD8 lymphocytes. More typical for adenocarcinoma was mature B-cell reaction (CD20). Effector (perforin-positive) populations of lymphocytes also were related to histological type of cancer: for adenocarcinoma presence of CD4-positive cytotoxic lymphocytes and CD27-expression on effector cells. Perforin-containing lymphoid cells were in opposite correlation to erythrocaryocytes. Conclusion Subpopulational lymphocyte content of bone marrow is related to histological variant of cancer and erythropoiesis in lung cancer patients.

PSVI-22 Immunity indicators of calves obtained from cows with ketosis during the treatment process

Abstract The purpose of these studies was to determine the indicators of cellular and humoral immunity of calves. Compared to calves obtained from untreated cows, the number of T-lymphocytes in young calves born from cows treated with Timolin increased the level of T-cells to 32.9%, while using Timosplenin 45%. As a result of the use of immunomodulators in treating cows with ketosis in mothers, the relative content of T-lymphocytes in the blood of calves born from them increased when using the drug Leucogen by 1.7 times, Timosplenin by 2.3 times, Timalin in 2.2 times. The use of Leucogen for cows with ketosis contributed to an increase in the percentage of B cells in the blood of calves born by 23%, Timosplenin 83%, and Timalin 42%. At the same time, the absolute number of B-lymphocytes in the treatment of “Leucogen” increased 1.9 times and reached the level of healthy calves, in the treatment with the preparations “Timosplenin” and “Timalin” - the increase was, respectively, 3.0 and 2.3 times and was higher this indicator in young animals born from healthy calves. Thus, the inclusion of dry immunomodulators in the treatment regimen of patients with ketosis leads not only to normalization of metabolism, but is also accompanied by an increase in the level of cellular immunity factors in calves born from them. Under the influence of “Leucogen,” the percentage of lymphocytes was normalized, whereas with the introduction of “Timalin” and “Timosplenin,” the content of this type of cells in newborn calves even exceeded those in healthy young.

Comparison of Gold Standard Genescan with NGS-Based TCR-Beta Clonality Analysis Using Oncomine TCR Beta-Short Read Assay

Introduction All mature T-cell-based lymphoid malignancies harbor identical (clonal) rearrangements of their T cell receptor (TCR) genes (van Dongen et al. Clinica Chimica Acta. 1991, 198, 1-92). The clonality assessment of the rearranged TCR gene is particularly important for the identification, characterization and monitoring of T-cell neoplasms since histology and immunophenotyping alone is not enough to make conclusive diagnosis in all cases. Diagnostic clonality testing is currently based on the parallel analysis of the rearranged TCR-gamma and TCR-beta chain genes. This is done using a multiplex PCR developed within the European BIOMED-2/EuroClonality consortium (van Dongen et al. Leukemia. 2003, 17, 2257-2317) followed by capillary electrophoresis of the resultant PCR products. Although, this method is considered the "gold standard", which is established worldwide in many molecular diagnostic laboratories, the resolution of this approach is limited, especially in cases with low percentage of clonally rearranged TCRs and a high mixture of non-clonally rearranged T-cells. Next generation sequencing (NGS) is a powerful tool to provide resolution at single gene level. This also holds true for NGS assays, which are able to detect all unique TCR rearrangements in a given sample with very high resolution and sensitivity. This approach can be employed for precise assessment of the immune repertoire, minimal residue disease and T-cell clonality. Detailed insights into the clonotypes provides a great potential for early diagnosis of T-cell neoplasms, and the identification of individual clones is essential for monitoring of the disease. Methods We investigated 19 formalin-fixed paraffin-embedded (FFPE) tissue samples from celiac disease patients (n = 14) and routine diagnostic cases suspicious of lymphoma (n = 5). After DNA extraction, we performed multiplex PCRs using the BIOMED-2 TCR-beta primer sets (van Dongen et al. Leukemia. 2003, 17, 2257-2317) followed by capillary electrophoresis. For TCR-beta NGS, the Oncomine™ TCR Beta-SR DNA Assay (Thermo Fisher Scientific) was used according to the manufacturer's instructions and sequencing was performed on the IonTorrent S5. Identification of individual clonotypes and bioinformatics analysis of the data was done with the help of the IonReporter software (Thermofisher Scientific). It is worth noting that the 14 celiac disease samples were previously analysed by a different TCR-beta NGS approach (Ritter et al. Gut. 2018, 67, 644-653) and utilized for the comparison of the NGS-data. Results 17 out of 19 cases analysed by TCR-beta multiplex PCR (BioMed-2) followed by capillary electrophoresis and by the Oncomine™ TCR Beta-SR assay displayed a very similar length distribution of the PCR products. This holds true for samples with a clonal appearance and for samples with an oligo-/polyclonal pattern. In two discrepant cases, the Primerset B of the BioMed-2 approach showed a dominant amplification product, which was not as clear by TCR-beta NGS in which both cases displayed an oligoclonal TCR-beta gene rearrangement pattern with a few dominant T-cell populations. Both samples were from celiac disease patients, which mainly present a pronounced oligoclonal TCR-beta gene rearrangement pattern. Strikingly, the NGS data from the Oncomine™ TCR Beta-SR DNA Assay delivered highly comparable results when compared to the sequencing data of Ritter et al. 2018 despite having completely different primer sets and a different NGS platform. Conclusion Our comparison of the conventional multiplex PCR (BioMed-2) and TCR-beta NGS (Oncomine™ TCR Beta-SR DNA Assay) demonstrated a very high concordance (17/19 cases) of the molecular data. The two discordant cases can be explained by an over-interpretation of dominant species BioMed-2 Primerset B amplifications, which often show up in cases with low T-cell content or oligoclonal T-cell counts. These T-cell clonotypes are detectable by TCR-beta NGS only at a low percentage because of combination of all type of rearrangements in one assay. The robustness and reliability of NGS-based TCR-beta clonality testing was demonstrated by comparison of two completely different assays, leading to very similar results for all celiac disease patients. We are thus very confident that NGS-based clonality testing will be the "gold standard" of the future. Disclosures Looney: Thermo Fisher Scientific: Employment. Allen:Thermo Fisher Scientific: Employment.

The influence of chemical environmental factors on functions and interrelationships of regulatory systems in children

In children under aerotechnogenic impact of phenol, methanol and formaldehyde, these substances are identified in the blood in significantly higher concentrations than in the unexposed group. There was found the activation of the mechanisms of the innate immunity (phagocytic activity) and inhibition of markers of the acquired immunity (reduction of relative indices of the content of T-lymphocytes (CD3+) and their subsets (CD4+- and CD8+-lymphocytes). Out of the markers of neuroendocrine regulation there was proved the significant decline of cortisol. Under the impact of anthropogenic chemical environmental factors there are occurred rearrangement and the transformation of interactions within the neuroendocrine and immune systems, as well as “early”, not manifested yet in the nature of specific nosological forms, but over time having the possibility to lead to the shaping of various pathology changes in the neuroendocrine regulation of the immune system.

Immune Desertic Landscapes in Hepatocellular Carcinoma Shaped by β-Catenin Activation.

About one third of cases of hepatocellular carcinoma (HCC) show gain-of-function mutations of CTNNB1 (β-catenin) that correlate with sparse intratumoral T-cell content, as observed previously in an ample spectrum of malignancies, and there is mounting preliminary evidence that such HCC cases are refractory to treatment with PD-1 checkpoint inhibitors. Elegant hepatocarcinogenesis experiments by in vivo gene transfer to mouse hepatocytes show that coexpression of active forms of β-catenin result in poor T-cell infiltrates, faster progression in immunocompetent hosts, and unresponsiveness to immunotherapy with checkpoint inhibitors.See related article by Ruiz de Galarreta et al., p. 1124.