Subpopulation Composition Of Lymphocytes Research Articles

Minor population of NK lymphocytes with CD19 coexpression

Single reports were published concerning a minor subpopulation of NK cells with weak coexpression of the B cell antigen CD19 in the patients’ blood and bone marrow. The frequency and relative number of CD56+CD19+dim cells is virtually not assessed, and there is no data on their phenotypic characteristics, as well as the connection of this subpopulation with any disease state. The purpose of the present study was to assess the frequency, relative quantity and phenotypic characteristics of CD56+CD19+dim lymphocytes in blood of patients referred for assessment of the lymphocyte subpopulation profile. Peripheral blood of immunocompromised individuals was taken, and subpopulation composition of lymphocytes was determined using eight-color flow cytometry (markers: CD3, CD4, CD8, CD19, CD25, CD45, CD56, HLA-DR). To estimate incidence of the CD56+CD19+dim subpopulation, we have carried out a retrospective analysis of LMD files on 1210 studies for 935 patients (average age, 39.8±14.7 years old) including 84 children under 18 years old. The study was performed repeatedly for some patients. Phenotyping of CD56+CD19+dim cells was performed using a panel of antibodies to B cell, T/NK cell antigens. The occurrence of blood samples containing CD56+CD19+dim was 1.2%, with a relative content of 2.1±1.9% among total lymphocyte population (0.8±0.6% of leukocytes). Long-term persistence of the subpopulation was noted in the patients throughout the entire observation period. The comparison of specific marker expression by NK CD56+CD19+dim, and CD56+CD19- cells revealed high expression of CD2, CD57, reduced expression density of CD7, CD16, CD38. The phenotype of the studied NK cell subpopulation was as follows: CD56+dimCD19+dimCD2+brightCD7+dimCD11c+CD16+dimCD38+dimCD45RA+CD57+CD94+dimNKG2D+CD3-CD4-CD5-CD20-CD21-CD25-CD45R0-CD62L-CD79b-CD117-, with variable expression of CD8 and HLA-DR. The phenotype is consistent with activated terminally differentiated adaptive NK associated with cytomegalovirus infection. The individuals with CD56+CD19+dim had a history of CMV-infection and reactivation of chronic EBV-infection at the time of the study. A probable cause of CD19 coexpression may be trogocytosis of B cell membrane fragments by natural killer cells during active EBV-infection. CD56+CD19+dim lymphocytes can reach noticeable values thus altering the results of studies performed by flow cytometry. The errors are most likely to occur upon assessing the minimal residual disease levels in acute B cell leukemias. The minor CD56+CD19+dimNK subpopulation may be detected in routine immunological analysis. Its functional features and association with certain disorders require further studies.

Lymphocytes and monocytes subpopulation composition in peripheral blood in patients with atrial fibrillation and futter

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): NSC "Institute of cardiology, clinical and regenerative medicine named by acad. Strazhesko" NAMS of Ukraine The purpose of the trial is to compare the state of intracardiac and systemic hemodynamics in patients with paroxysmal and persistent forms of atrial fibrillation and flutter that appeared on the base of arterial hypertension, depending on the lymphocytes and monocytes subpopulation composition in peripheral blood. Materials and methods The study involved 103 patients with atrial fibrillation and flutter and were divided into three main groups: I – with paroxysmal form of atrial fibrillation, II – with a persistent form of atrial fibrillation, III – with a persistent form of atrial flutter. The subpopulation composition of lymphocytes and monocytes in peripheral blood was assessed by the method of flow cytometry. Hemodynamic parameters were evaluated using echocardiography, office blood pressure measurement, daily blood pressure monitoring, and transesophageal echocardiography. For control, the data of patients with hypertension but without the above-mentioned arrhythmias were used, and practically healthy people who entered groups IV and V, respectively. Results and discussion The number of cells with cytotoxic activity (NK and T-NK) in both absolute and percentage values was significantly higher in groups with arrhythmia than in practically healthy individuals. A mathematically significant decrease of T-regulatory cells number (p ≤0.05) in the second and third groups compared to the control groups was detected, same as increased number of classical and intermediate monocytes fractions. Comparing echocardiogram results indicators between groups I, II, III and I V, a mathematical significant difference (p≤0.05) between the LVID was revealed: LVIDd, LVEDV, LVESV, IVS, PW thickness; LA AP, RVmid diameter at end-diastole, E/A and LVMI. Compared to V the group difference was significant only between LVIDd, LA AP size, IVS thickness, and PW thikness. Office systolic blood pressure was significantly higher in patients with arrhythmias. According to daily blood pressure monitoring, the difference between the indicators of average and maximum diastolic pressure was significant. Conclusions In patients with atrial fibrillation and flutter as a result of hypertension, compared to patients without arrhythmias or healthy people, there is an increased content of pro-inflammatory monocytes subpopulation, T-cytotoxic cells and a decreased amount of T-regulatory cells. According to EchoCG, these patients had more severe structural changes in the myocardium: an increased LA AP and LV hypertrophy, a thickening of IVS and PW, and diastolic dysfunction. The recorded measurement indicators of SBP and DBP differed mathematically significantly, which indicates hemodynamic disorders caused by both rhythm disturbances and manifestations of arterial hypertension.

Половые различия в субпопуляционном составе лимфоцитов периферической крови при язвенном колите отражают специфические особенности иммунопатогенеза воспалительных заболеваний кишечника у мужчин и женщин

Objective. To identify the features of the composition of peripheral blood lymphocyte subsets in women with ulcerative colitis (UC). Patients and methods. The study included 32 women: 21 patients with UC and 11 healthy volunteers (control group) without inflammatory bowel disease (IBD). The major subsets of T and B lymphocytes were detected by multicolor flow cytometry. Results. The analysis of peripheral blood CD3+ T cell differentiation in women with UC in comparison with healthy controls showed that the Tcyt group had a significant decrease in naive Tcyt cells with an increase in the level of Tcyt cell subsets with pronounced effector properties (EM Tcyt and TEMRA Tcyt). In the subsets of central and effector memory Th cells in women with UC compared to healthy controls, Th17.1-CM cells were significantly different among central memory (CM) cells, and Th17-EM and Th17.1-EM cells were significantly different among effector memory (EM) cells. No significant differences were found in any of the Th17 cell subsets, but there was a tendency towards decreased levels of Th17.1 cells. The analysis of the subsets of T follicular helper (Tfh) cells revealed significant changes only in the level of Tfh17/Tfh22-EM cells (Tfh17-EM cell subset). No significant differences in the B cells were revealed, except for plasmocytes with IgD–CD27++ phenotype, which were increased in UC patients. Conclusion. Changes in peripheral blood T- and B-lymphocytes in women with UC can reflect not only activation of the peripheral immune system, but also induction of immunoglobulin production by B cells activated by T follicular helper cells. For the first time, specific differences between women and men in the total pool of Th17/Th22 cells, “double-positive” Th17, Tfh2 and Tfh17/Tfh22 cells were identified. In contrast to men, no manifestations of a Th2-specific response at the cellular level were observed in women with UC. The revealed sex-related differences in the composition of lymphocyte subpopulations in peripheral blood in UC reflect specific features of the IBD immunopathogenesis in men and women and can be considered in the management of patients with IBD. Key words: inflammatory bowel disease, ulcerative colitis, T helper cell subsets, B lymphocytes, sex-related differences

Features of hematological and immune disorders in the combined treatment of breast cancer patients with the risk of development of late radiation damage

Background. Breast cancer (BC) is one of the most common forms of malignant neoplasms among the female population of Ukraine. The widespread use of radiation therapy in the treatment of BC, along with the improvement of treatment efficiency, inevitably leads to an increase in the probability of early and/or late radiation injuries (LRI), which puts before scientists the task of a detailed study of this problem and the search for ways to prevent the development of LRI.The body’s reaction to the development of a malignant neoplasm and to antitumor treatment is largely determined by the state of homeostatic mechanisms, in the regulation of which one of the key roles is played by the immune system. Recently, special attention has been focused on the role of immune inflammatory responses in the pathogenesis of LRI in cancer patients. All of the above determines the urgency of finding a differential approach to immunocorrective therapy as a prevention of the development of LRI. Рurpose – to determine the characteristics of changes in hematological indicators and the subpopulation composition of lymphocytes during immunocorrective therapy as a prevention of the development of LRI in patients with BC. Materials and Methods. 55 patients with BC were examined. The patients were divided into groups: the comparison group (n=13) – patients with BC with the risk of developing LRI who were given standard therapy, the main group (n=15) – patients with BC with the risk of developing LRI who were given immunocorrective therapy against the background of standard treatment. The control group (n=27) consisted of patients with BC without the risk of developing LRI. The groups were comparable in terms of age and disease stage. Results. Immunocorrective therapy increased the number of erythrocytes, hemoglobin level, CD19+-, CD3+CD8+-lymphocytes, NK-cells, CRP, IL-6, IL-2 and TNF-α in patients of the main group. In patients of the comparison group, after treatment, lower levels of erythrocytes, hemoglobin, lymphocytes, the relative number of CD8+-T-lymphocytes, CD19+-B-lymphocytes and higher levels of the relative number of eosinophils, monocytes, neutrophils, cytokine levels, and CRP were found in comparison with the indicators of patients, who underwent immunocorrective treatment. In patients with BC of the main group, immunocorrection reduces systemic changes that can contribute to the development of late radiation damage and recurrence of the oncological process. The inclusion of immunocorrective therapy with the use of melatonin had a positive effect on the hematological and immune indicators of patients. Conclusions. Immunocorrective therapy in patients at risk of developing LRI optimized immune and hematological parameters: it contributed to an increase in the number of erythrocytes by 1.25 times, hemoglobin level by 1.6 times, CD19+- (1.5 times), CD3+CD8+-lymphocytes (1.1 times), and a decrease in NK cells (1.77 times), CRP by 2.19 times, IL-6 by 1.8 times, IL-2 by 2.13 times, and TNF-α by 3 times, 22 times. It was found that in patients with BC with the possibility of developing LRIwithout immunotherapy, lower levels of erythrocytes (by 1.15 times), hemoglobin (by 1.13 times), lymphocytes (by 1.3 times), and the relative number of CD8+ were observed after treatment - T lymphocytes (1.4 times), CD19+ B lymphocytes (6.5 times) and higher levels of relative numbers of eosinophils, monocytes, neutrophils, cytokine levels (1.5 times) and CRP (2.1 times) in comparison with the indicators of patients who underwent immunocorrective treatment. The appointment of immunocorrective therapy with melatonin in a daily dose of 9 mg during radiation treatment for patients with BC with the risk of developing LRI led to the normalization of hematoimmune indicators, a significant decrease in the systemic inflammatory reaction.

Hematological and immune disorders in colorectal cancer patients with liver metastases after radiofrequency ablation

Background. Colorectal cancer (CRC) is one of the most common malignant neoplasms in the world. It ranks third in the structure of oncological morbidity and second in the structure of mortality. The liver is the most common site of metastatic spread of CR and 14 to 18% of patients have liver metastases at diagnosis. Radiofrequency ablation is currently the most promising method of treating metastatic lesions.
 Рurpose – to study quantitative changes in hematoimmunological indicators and their influence on antitumor reactivity in patients with colorectal cancer after RFA of liver metastases.
 Materials and Methods. Clinical and laboratory examination was carried out in 12 patients with colorectal cancer with metastases in the liver, the majority of patients were over 60 years old. Adenocarcinoma was histologically determined in all patients, and most of them had a moderate degree of malignancy (G2). The study was carried out in three stages: I – one day before radiofrequency ablation (RFA), II – 3 days after RFA, III – 14 days after RFA of liver metastases.
 Results. Immune and hematological indicators of the development of the inflammatory response after RFA in patients with colorectal cancer with liver metastases were established. On the 3rd day (II stage) after RFA, a significant increase in the total number of leukocytes, the ratio of neutrophils to lymphocytes, the number of eosinophils, a violation of the balance of CD4+ and CD8+ lymphocytes, a decrease in the number of NK- and NKT-cells, an increase in the percentage of CD3+ HLA-Dr -, CD4 +PD1+, CD8+PD1+ lymphocytes. On the 14th day (stage III), most of the parameters were close to those determined before the treatment, except for the reduced number of NK and NKT cells and the increase in the level of platelets.
 Conclusions. A number of hematological changes on the 3rd day after RFA related to the inflammatory reaction were identified: an increase in the total number of leukocytes, an increase in the ratio of neutrophils/lymphocytes from 1.72 to 4.12, a significant decrease in the relative and absolute number of eosinophils. But on the 14th day, after the inflammatory reaction subsided, the normalization of most of the studied indicators was observed. A violation of the subpopulation composition of lymphocytes was established in patients with CR metastases in the liver, on the 3rd day after RFA. Those that had taken place even before the intervention (low number of CD8+ lymphocytes and NK cells) and additional ones appeared (increased CD4+/CD8+ ratio, increased percentage of CD3+ cells HLA-Dr+, CD4+ and CD8+ lymphocytes PD1+ (CD279+), decrease in the number of NKT cells). On the 14th day, the picture approached the initial one, with the exception of the number of NK and NKT cells. We believe that timely correction of inflammatory immunosuppression in the early days after RFA can shorten the period of vulnerability to recurrence of CR, and in the long term potentiate the positive effect of RFA on antitumor reactivity.

Assessment of neutrophil degranulation intensity and changes in neutrophil phenotype by FCᵧRIIIB expression level in blood of patients with COVID-19 and convalescents

Introduction. Disease severity in hospitalized COVID-19 patients correlates with the relative content in the blood of a specific low-density neutrophilic granulocyte (NG) population, whose cells are characterized by reduced granularity, high heterogeneity in the expression of FcᵧRIIIb (CD16) and a tendency to spontaneous autolysis (netosis).The aim of the study was to compare the intensity of NG degranulation and the FcᵧRIIIb expression by these cells in blood of patients with COVID-19 and convalescents.Materials and methods. The blood leukocytes of 40 patients diagnosed with COVID-19, 33 convalescents and 20 healthy donors (control) were examined using flow cytometry. To identify NG (CD16+-granulocytes) and to assess the surface expression of the netosis molecular trigger (FcᵧRIIIb), a single-color reagent of labeled monoclonal antibodies CD16-FITC was used. Immunophenotyping of lymphocytes was performed using two four-color reagents. In addition, cell debris content was determined in each Lyse/No-Wash-stained whole blood sample. The presence of neutrophils at the stage of netosis was confirmed by microscopy. Cytokine production was determined on an automatic enzyme immunoassay analyzer.Results. Against the background of characteristic changes in the lymphocyte subpopulation composition and cytokine production, in blood of COVID-19 patients with acute pneumonia, hypoxia and tachycardia a more intense degranulation of NG (2.6 times), higher degree of CD16 expression heterogeneity (1.8 times) and an increased leukocytolysis intensity (1.6 times) were observed compared to convalescents who have undergone severe and moderate forms of the disease. In convalescents, the degree of differences of these indicators compared to control values varied in concordance with the disease severity.Conclusion. The nature of changes in the parameters studied in COVID-19 patients in the acute phase of the disease and during the period of convalescence confirms the possible mechanism of the development of complications due to an imbalance between the activity of elastase released from NG granules and its plasma inhibitor α1 -antitrypsin.

Clinical and immunological features of the combined course of pertussis and rhinovirus infection in children

The association of pertussis with various respiratory infections in children is the leading factor determining the complicated course and unfavorable outcome of the disease.Objective. To analyze clinical and immunological features of the combined course of pertussis and rhinovirus infection.Children characteristics and research methods. The authors observed 20 patients: 10 (50%) children were under the age of 1 year, 5 (25%) children of 1–3 years old, 3 (15%) children of 4–6 years, 2 (10%) children of 7 -14 years old.Results. The rhinovirus infection developed mainly at 1-2 weeks of illness in 18 (90%) children. Bronchitis developed in 11 (55%) children, pneumonia – in 4 (20%). Special attention was drawn to the low content of NK cells in 82.4% of patients when assessing the subpopulation composition of lymphocytes. The cytokine profile was characterized by a low level of interferon-gamma and interferon-alpha production – in 94.4 and 61.1% of patients, respectively.Conclusion. The combination of pertussis and rhinovirus infection in children contributes to the uneven course of the disease, the frequent development of bronchopulmonary complications.

Efficiency of human plague vaccination in tuvinian natural plague focus. Message 2: dynamics of immune status indicators after revaccination

Relevance. In Russia, the live plague vaccine (LPV) is used for specific prophylaxis of plague. Immunological monitoring of humans vaccinated by LPV in order to search for informative diagnostic markers, as well as to improve the tactics of epidemiological surveillance of plague enzootic territories is an urgent area of research.The aim is to assess the parameters of cellular and humoral immunity in humans revaccinated by LPV who permanently reside on territory of the Tuvinian natural plague focus.Materials and methods. The study involved 76 volunteers from the Republic of Tuva, revaccinated by LPV. Blood sampling was performed before vaccination and 1, 3, and 6 months after revaccination. The study included the determination of cytokine production (IFN-γ, IL-4, TNF-α), specific antibodies, immunoglobulins (IgM, IgG, IgA and IgE) and lymphocyte subpopulation composition (CD3, CD4, CD8, CD16, CD19, immunoregulatory index).Results. A decrease in IgG and IgM and an increase in IgA were found after vaccination with LPV and their increase after revaccination. Correlation relationships were revealed between immunoglobulins, B cells and IL-4. Revaccination leads to an increase seroconversion. The activation of humoral immunity in humans vaccinated against plague is also evidenced by dynamics of changes in the subpopulation composition: an increase in B-lymphocytes and natural killer cells, a decrease in T-helpers and immunoregulatory index, and cellular immunity stimulation is an increase in spontaneous and induced production of pro- and anti-inflammatory cytokines.Conclusion. It has been shown that LPV is capable of causing the body’s immune restructuring and activating the cellular and humoral mechanisms of immunological protection. For a complete understanding of the development and preservation of antiplague immunity, it is necessary to continue the annual immunological monitoring of the population living on the territory of the Tuvinian natural plague focus, using additional modern research methods.

Markers of progression of liver fibrotic changes in patients with chronic toxic drug-induced hepatitis

Background. In recent years, as a result of the growing expansion of the pharmaceutical market, there has been a clear trend towards an increase in the incidence of chronic toxic drug-induced hepatitis of drug genesis (TDIH). The appearance of fibrosis is considered the most important histological change that determines the further course of the disease. Therefore, the search for non-invasive or minimally invasive markers for assessing fibrotic changes in the liver remains an urgent issue in clinical practice. The purpose was to determine the diagnostic value of immunological parameters for stratification of the severity of liver fibrosis in patients with TDIH. Materials and methods. The study included 41 patients with TDIH, who were divided into three groups: group I consisted of 12 people without liver fibrosis (F0), group II— 22 patients with moderate fibrosis (F1-F2), group III— 7 individuals with severe liver fibrosis (F3-F4). Shear wave elastography was performed using a Soneus P7 system (Ukraine-Switzerland). All patients underwent a biochemical blood test with the determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST). The subpopulation composition of lymphocytes, circulating immune complexes (CIC), the level of interleukins (IL-6, IL-10) and tumor necrosis factor α were assessed. Results.The progression of liver fibrosis is accompanied by an increase in cytolytic syndrome: patients with severe fibrosis have a 3.3-fold increase in the ALT (p<0.05) compared to the controls and a 2.1-fold (p<0.05) compared to that in patients with moderate fibrosis. The AST level is significantly higher— by 4.6 times (p=0.023) in patients with severe fibrosis than in those with moderate fibrosis. With the progression of liver fibrosis, there is a significant decrease in cellular immunity, an increase in the level of CIC and pro-inflammatory cytokines with a simultaneous decrease in the content of anti-inflammatory cytokines, which is confirmed by correlations between the liver stiffness index according to shear wave elastography data and the level of T-helpers (r=–0.466; p=0.03), IL-6 (r=0.364; p=0.01), IL-10 (r=–0.331; p=0.039) and CIC (r=0.381; p=0.017). Conclusions. Markers of the diagnosis of severe liver fibrosis in patients with TDIH are indicators such as the ratio of IL-6/IL-10 higher than 0.83 (sensitivity 81.8%, specificity 78.9%), CIC level more than 4.3 optical density units (sensitivity 77.3%, specificity 72.2%), the ratio of T-helpers/T-suppressors is less than or equal to 1.6 (sensitivity 72.7%, specificity 57.9%).

Comparison of the composition of lymphocyte subpopulations in non-relapse and relapse patients with squamous cell carcinoma of the head and neck before, during radiochemotherapy and in the follow-up period: a multicenter prospective study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG)

BackgroundRadiochemotherapy (RCT) has been shown to induce changes in immune cell homeostasis which might affect antitumor immune responses. In the present study, we aimed to compare the composition and kinetics of major lymphocyte subsets in the periphery of patients with non-locoregional recurrent (n = 23) and locoregional recurrent (n = 9) squamous cell carcinoma of the head and neck (SCCHN) upon primary RCT.MethodsEDTA-blood of non-locoregional recurrent SCCHN patients was collected before (t0), after application of 20–30 Gy (t1), in the follow-up period 3 (t2) and 6 months (t3) after RCT. In patients with locoregional recurrence blood samples were taken at t0, t1, t2 and at the time of recurrence (t5). EDTA-blood of age-related, healthy volunteers (n = 22) served as a control (Ctrl). Major lymphocyte subpopulations were phenotyped by multiparameter flow cytometry.ResultsPatients with non-recurrent SCCHN had significantly lower proportions of CD19+ B cells compared to healthy individuals before start of any therapy (t0) that dropped further until 3 months after RCT (t2), but reached initial levels 6 months after RCT (t3). The proportion of CD3+ T and CD3+/CD4+ T helper cells continuously decreased between t0 and t3, whereas that of CD8+ cytotoxic T cells and CD3+/CD56+ NK-like T cells (NKT) gradually increased in the same period of time in non-recurrent patients. The percentage of CD4+/CD25+/FoxP3+ regulatory T cells (Tregs) decreased directly after RCT, but increased above initial levels in the follow-up period 3 (t2) and 6 (t3) months after RCT. Patients with locoregional recurrence showed similar trends with respect to B, T cells and Tregs between t0 and t5. CD4+ T helper cells remained stably low between t0 and t5 in patients with locoregional recurrence compared to Ctrl. NKT/NK cell subsets (CD56+/CD69+, CD3−/CD56+, CD3−/CD94+, CD3−/NKG2D+, CD3−/NKp30+, CD3−/NKp46+) increased continuously up to 6 months after RCT (t0-t3) in patients without locoregional recurrence, whereas in patients with locoregional recurrence, these subsets remained stably low until time of recurrence (t5).ConclusionMonitoring the kinetics of lymphocyte subpopulations especially activatory NK cells before and after RCT might provide a clue with respect to the development of an early locoregional recurrence in patients with SCCHN. However, studies with larger patient cohorts are needed.Trial registrationObservational Study on Biomarkers in Head and Neck Cancer (HNprädBio), NCT02059668. Registered on 11 February 2014, https://clinicaltrials.gov/ct2/show/NCT02059668.

Biological activity of 4-ethynyl-, 4-oxy-, 4-butoxypropylpyperidine and azaheterocyclic compounds

The new chemical series of compounds is promising for the search new effective immunostimulating drugs. Acute toxicity and the effect on peripheral blood hemogram parameters and on the subpopulation composition of lymphocytes were determined in the compounds (piperidine-containing derivatives): 1-(3-n-butoxypropyl)-piperidine-3-methyl-4-spiro-5'-imidazolidine-2',4'-dione,1-(2-ethoxyethyl)-4-carboxy-4-hydroxypiperidine, bis[1-(2-ethoxyethyl)-4-ethynyl-4-hydroxypiperidine) dihydrochloride and, 1:1 complexes of β-cyclodextrin with 1-(2-ethoxyethyl)-4-(2-ethoxyethoxyl)-4-ethynylpiperidine, 1-(2-ethoxyethyl)-4-(2-methoxyethoxyl)-4-ethynylpiperidine, 7-(2-methoxyethyl)-2,2-dimethyl-3-thia-7-azabicyclo[3.3.1]nonane or 3-(2-morpholinoethyl)-7-(3-isopropoxypropyl) diazabicyclo[3.3.1] nonane with β-cyclodextrin. Compound ˗ 1-(3-n-butoxypropyl)piperidine-3-methyl-4-spiro-5'-imidazolidine-2',4'-dione, exceeds the immunostimulating activity of the comparison drug of levamizole by 3,1 times and has low toxicity.

Composition of lymphocyte subpopulations in normal and mildly reactive peripheral lymph nodes in cats.

Flow cytometric (FCM) immunophenotyping of lymphoid tissue aspirates is an available adjunct for feline lymphoma diagnostics. Reference data have only been established for feline peripheral blood. Studies investigating the composition of normal and mildly reactive feline lymph nodes (LNs) are lacking. The aim of this prospective study was to establish reference data for lymphocyte subpopulations in normal and mildly reactive feline peripheral LNs using a standardised multicolour panel of antibodies. Macroscopically inconspicuous mandibular and/or popliteal LNs from 31 adult cats, which were euthanased for reasons other than haematological diseases, were excised and processed within 5 h after death. Multicolour flow cytometry using eight different feline-specific, anti-canine and human cross-reactive monoclonal antibodies used in current diagnostic marker panels was performed after cytological exclusion of pathological states and complemented by lymphocyte clonality testing, histopathology and immunohistochemistry (IHC) to ensure the absence of lymphoid disease. Of 31 cats, the immunophenotyping data of 24 individuals could be included as histopathology and clonality testing excluded a pathological condition. Lymphocyte populations showed the following positive antibody reactions: CD18+ 86.3% ± 13.86%, CD3+ 54.81% ± 11.10%, CD5+ 57.39% ± 12.66%, CD21+ 40.42% ± 12.40%, CD79alphacy+ (CD79αcy) 30.41% ± 13.49% and CD14+ 0.75% ± 1.35%. There were 30.88% ± 13.48% CD4+ and 12.91% ± 6.68% CD8+ cells. Cytology revealed a mixed population of mostly lymphoid cells in all samples. The absence of a monoclonal/oligoclonal neoplastic population was confirmed by lymphocyte clonality testing. Histopathology and IHC showed a normal or mildly reactive pattern in all cases. This study establishes FCM immunophenotyping data of lymphocyte populations of normal and mildly reactive feline peripheral LNs. For the first time, anti-CD5, CD4, CD8 and CD21 reference data in normal and mildly reactive feline peripheral LNs are presented. CD18, CD3, CD14 and CD79αcy have been used to establish reference data for the first time in any feline material.

The State of Systemic and Local Immunity with Non ? Specific Vulvovaginitis in Adolescents

The study showed that there is a secondary immune deficiency in the subacute course, and in acute immune deficiency it was not detected. In acute course compared with subacute one, there is an increased production of IFN-γ, which indicates the direct dependence of the pro-inflammatory cytokines activity level on the clinical course of the inflammatory process. A study of vaginal secretion phagocytosis revealed that in acute cases, the phagocytic function of the local secretion is satisfactory, while in the subacute case, satisfactory absorption function decreases the bactericidal activity of the vaginal secretion, which indicates the need for correction of the phagocytic link. The use of Roncoleukin parenterally in acute course does not affect normal immune status indicators, i.e. has a modulating effect depending on the initial immune status. In subacute case, local application leads to an increase in the absorption and oxygen-dependent bactericidal ability of the vaginal secretion, which contributes to the rapid regression of the clinical manifestations of vulvovaginitis, parenterally leading to normalization of the main indicators of the subpopulation composition of lymphocytes.

Assessment of the population and subpopulation composition of lymphocytes and their relationship in patients with sensorineural hearing loss

Phenotyping of blood lymphocytes (CD3+, CD4+, CD8+, CD9+, CD16+, CD20+, CD21+, CD23+, CD25+, CD95+) was performed by indirect immunofluorescence. Individuals with sensorineural hearing loss were found to have hyperactivation in both T- and B-cell links immunity. The revealed changes indicate a violation of immunoregulation through factors of intercellular cooperation, and the established relationships between populations and subpopulations of lymphocytes indicate increased activity components of the immune system, their important role in the implementation of the immune response in persons with sensorineural hearing loss.

Features of lymphocyte differentiation in ANCA-associated and virus-associated glomerulonephritis in children

Glomerulonephritis is a heterogeneous group of immuno-inflammatory diseases. The purpose of the study: to analyze the subpopulation composition of lymphocytes in children with various types of glomerulonephritis. 50 children aged 1 to 16 years were examined: 1 group — 17 children aged 1 to 16 years with ANCA-associated glomerulonephritis; 2 group — 33 patients aged 2 to 16 years with virus-associated glomerulonephritis. Diagnostics of the subpopulation composition of lymphocytes was performed by flow cytometry using non-flushing technology using a three-color combination of monoclonal antibodies. It was found that the development of ANCA-associated glomerulonephritis is accompanied by signs of immunodeficiency against the background of a relative increase in the content of T-lymphocyte subpopulations. Differentiation of lymphocytes in children with virus-associated glomerulonephritis is ambiguous: the process goes either along the path of activation of antiviral protection, or along the path of development of insufficient antiviral activity of immunocytes. In children with virus-associated glomerulonephritis, a decrease in the level of B-lymphocytes was found, correlating with hypoimmunoglobulinemia A, which is an additional factor in reducing resistance on the mucous membranes. The study of lymphocyte differentiation in the development of glomerulonephritis is important for identifying the main immunological mechanisms involved in the pathogenesis of these diseases.

Immune disorders after combined treatment of obese patients with endometrial cancer

Background. Immune disorders in antitumor treatment of obese patients may depend on the severity of the inflammatory process, which justifies the need to take into account the intensity of the latter in the analysis of complications of antitumor treatment in obese individuals. Purpose – evaluation of immune disorders after radiation therapy in obese patients with different intensity of the inflammatory process. Materials and methods. 65 patients with stage I–II endometrial cancer underwent pangysterectomy and a postoperative course of remote gamma therapy. Examinations were performed before treatment and after radiation therapy. Subpopulation composition of lymphocytes, phagocytic activity of neutrophils, levels of circulating immune complexes and immunoglobulins G, A, M were determined by standard methods. The SF-3000 «SYSMEX» analyzer was used to evaluate hematological parameters. The level of C-reactive protein (CRP) was determined using an analyzer «RESPONS 910». Insulin and leptin levels were assessed by enzyme-linked immunosorbent assay. Results and discussion. Immune and hematological parameters were evaluated in 3 groups of patients. Group I included patients with normal body weight (BMI < 25), II and III groups consisted of obese patients (BMI > 30). Group II included patients with a CRP level below the median, which was 18,0 mg/l, group III – patients with a CRP level above 18.0 mg/l. In patients of group II there was a higher absolute and relative number of lymphocytes, the absolute number of CD3+-, CD4+- and CD8+-lymphocytes compared with those registered in groups I and III. After radiation therapy, the absolute number of lymphocytes and lymphocyte subpopulations decreased in all groups. As before treatment, higher levels of these indicators were registered in group II. In obese patients, the relative number of NK cells was lower than that determined in patients of group I before and after treatment. Conclusions. Patients with obesity and low intensity of the inflammatory process have less pronounced immune disorders after radiation therapy compared with patients who had a normal body weight and individuals with obesity and high levels of CRP.

Major and minor lymphocytes subpopulations in peripheral blood and cerebrospinal fluid of children with meningitis

Introduction. The analysis of current publications indicates at our insufficient understanding of subpopulation composition of lymphocytes in peripheral blood and cerebrospinal fluid (CSF) during pediatric neuroinfectious diseases. It has been found that the main lymphocyte populations are divided into many small (minor) subpopulations.The purpose of this research was to assess percentage of major and minor blood and CSF lymphocyte subsets in children with aseptic viral meningitis (AM) or bacterial purulent meningitis (BM).Materials and methods. Phenotyping of blood and CSF lymphocytes of children aged from 4 months to 17 years diagnosed with AM (n = 86) and BM (n = 39) was carried out by using flow cytometry. As a comparison group, we analyzed peripheral blood and CSF samples collected from children with acute respiratory viral infections (ARVIs) associated with syndrome of meningism (n = 27). There was evaluated percentage of the major cell subpopulations (CD3+ T-lymphocytes, T-helpers — CD3+CD4+ Th, cytotoxic T-lymphocytes — CD3+CD8+ CTL, natural killer cells — CD3-CD16+CD56+ NK, B-cells — CD3-CD19+), as well as minor lymphocyte subsets (double positive (DP) (CD3+CD4+CD8+), double negative (DN) (CD3+CD4-CD8-) T-cells, NKT (CD3+CD16+CD56+), CD3-CD8+ NK, CD3+CD8dim and CD3+CD8 8bright).Results. It was found that the acute period of BM and AM vs. the comparison group (ARVI) was characterized by significant differences in the blood and CSF composition of major and minor lymphocyte subsets. In particular, blood T-cells, Th, CTL, NK, NKT, DN, CD3-CD8+ NK, CD3+CD8bright and CD3+CD8dim dominated in parallel with significantly lowered B-cell frequency in AM vs. BM. In the CSF of children with AM, T-cells and Th prevailed, whereas count of B-cells and CD3-CD8+ NK was lower compared to those in BM. In addition, further differences were revealed in CSF and blood cell subset composition depending on nosological entity, while maintaining differences in some major and minor lymphocyte subpopulations lacked in the comparison group. Calculating the CSF/blood ratio for the major and minor lymphocyte subsets uncovered the prevalence for the majority of cell subpopulations (the coefficients ranged from 1.2 to 16.4) in the CSF of the comparison group (ARVI), except B-cells, NK and CD3-CD8+ NK (coefficients ranged from 0.07 to 0.31). AM and BM were featured with various changes in the CSF/blood ratio found for most of the studied subpopulations in the acute period as well as the recovery phase highlighted with characteristic traits for each nosological form.Conclusion. The data obtained indicate about finding specific features in the activation of systemic and intrathecal immune response during viral and bacterial meningitis in children, which may be used as an additional differential diagnostic criterion.

Experimental Evaluation of Application of the Vaccine Strain <i>Yersinia pestis</i> EV NIIEG in Combination with Immune-Modulators

Abstract. Objective of the work was to conduct a comparative assessment of the immune-modulating effect of the combined use of Yersinia pestis EV NIIEG vaccine strain with Polyoxidonium and Ingaron preparations on a BALB/c mouse model.Materials and methods. Mice of the BALB/c line were immunized subcutaneously with Yersinia pestis EV NIIEG culture at a dose of 2.5 104 m.c. (1st group), in combination with Ingaron at a dose of 150 IU (2nd group) or with Polyoxidonium at a dose of 4 мg (3rd group), the 4th group is intact mice. On days 3, 7, 21 and 90 after immunization, the subpopulation composition of lymphocytes, the production of mediators of the cellular response (INF-ɣ and IL-10), the titers of specific antibodies to the capsular antigen of plague microbe (F1), the nuclear apparatus of lymphocytes, and the nature of histological changes in the organs of mice were determined. Characterization of immunogenic (protective) activity of the combined use of Y. pestis EV NIIEG with immune-modulators against Y. pestis 231 in experiments on BALB/c mice was performed on the 21st day after immunization through determining the number of dead animals and their average life expectancy.Results and discussion. The combined administration of Y. pestis EV NIIEG vaccine strain with Polyoxidonium or Ingaron to experimental animals allowed us to establish differences in the response of the immune system of biomodels, due to the mechanism of action of a specific immune-modulator. It has been established that both Polyoxidonium and Ingaron combined with Y. pestis EV NIIEG enhance the response of immune-competent cells in experimental animals, contribute to the activation of the humoral response and the production of mediators of the cellular response, do not have a damaging effect on the tissue of the macroorganism. At the same time, the efficacy of using combined vaccination of Y. pestis EV NIIEG with immune-modulators in the inoculation test is confirmed for Polyoxidonium only.

ВЛИЯНИЕ КАРБОПЛАТИНА С КУКУРБИТ[7]УРИЛОМ НА СУБПОПУЛЯЦИОННЫЙ СОСТАВ И ПРОЛИФЕРАТИВНУЮ АКТИВНОСТЬ ЛИМФОЦИТОВ IN VITRO

It is known that the use of delivery systems can reduce the toxicity of platinum compounds without losing a high therapeutic effect. In this work, the immunotoxic effect of carboplatin when co-added with cucurbit[7]uril was investigated, including the effect on the subpopulation composition of lymphocytes and their proliferative activity in vitro.

THE RESULTS OF THE STUDY OF IMPORTANT CLINICAL ASPECTS OF TTV INFECTION: SPECTRUM OF MANIFESTATIONS, ASSOCIATION WITH MINOR IMMUNODEFICIENCIES, EFFICACY OF ARTESUNATE

Background. TT, or Torque teno virus, is a widespread population of DNA-containing simple virus from the Anelloviridae family that can cause both chronic hepatitis and extrahepatic lesions, but is still an under-studied pathogen that lacks effective antiviral drugs.
 Aim of the study: to study the spectrum of clinical phenotype, immune status and efficacy of artesunate in chronic reactivated TTV infection in humans.
 Materials and methods. In this retrospective clinical case study, we examined the results of case histories of 67 patients aged 19 to 52 years (36 men, 31 women) with reactivated TTV infection (PCR data from blood cells) who received artesunate therapy. 38 additional patients with reactivated TTV infection with similar age, gender distribution, and clinical picture who did not receive artesunate constituted the control group.
 Immunological study included the study of indicators of total blood count, subpopulation composition of lymphocytes using laser flow cytofluorimetry (cytofluorometer Epics Xl, USA) and indirect immunofluorescence method with monoclonal antibodies to CD (CD3+, CD3+ CD4+, CD3+CD8+, CD3-CD19+, CD3-CD16+CD56 +, CD3+CD16+ CD56+) (Beckman Coulter reagents, USA). Phagocytosis was evaluated according to a latex test to determine the index of phagocytosis, the number of active phagocytes and phagocytic blood capacity. Serum immunoglobulin concentrations of the major classes (M, G, A) were determined by Mancini simple radial immunodiffusion. Concentration of IgE, IgD and IgG subclasses (IgG1, IgG2, IgG3, IgG4) in serum was measured by enzyme-linked immunosorbent assay (VectorBEST, RF). Serum mannose binding lectin concentration and myeloperoxidase activity were determined by enzyme immunoassay.
 Within 1 month of therapy, artesunate was administered at a dose of 50 mg twice a day 1 time for 12 hours orally after meals, and for 2-3 months with insufficient effectiveness of the previous course - at a dose of 50 mg three times a day 1 time for 8 hours orally after food.
 Statistical analysis of information was performed using structural and comparative analyzes. Methods of variational statistics were applied with the calculation of the parametric index of the Student’s T-test with the index of confidence probability p and the nonparametric criterion of the number of signs Z by Urbach Yu.V.
 Results of the study and discussion. Hepatic lesions were reported in only 34%, while non-hepatic forms of the disease - in 66% of cases. Extrahepatic manifestations were determined by chronic fatigue syndrome (34%), neuropsychiatric symptoms associated with temporal mesial sclerosis (32%), mononucleosis-like syndrome (16%), vasculopathy of small cerebral vessels (14%), encephalitis (5% of cases) . All patients were immunocompromised individuals. Only one case was diagnosed with HIV infection, and all other patients suffered from minor immunodeficiencies, including deficiency of natural killer T-lymphocytes (49%), natural killer cells (30%), cytotoxic T-lymphocytes (24%), IgE and/or IgD, mannose-binding lectin (15%), myeloperoxidase (12%), IgA (4%), and idiopathic CD4+ T-cell lymphopenia (3% of cases). Artesunate therapy was effective in 62% of cases (p<0.05; Z<Z0.05), providing removal of virus DNA from blood cells according to PCR for 1-3 months. In 21% of cases there was a partial and 17% - complete resistance of the virus to artesunate. This is 10-15% more effective than previously reported in alpha interferon preparations, with better tolerability and ease of use for artesunate. According to PCR, the mean number of viral particles in blood cells during therapy decreased from 97 to 11 thousand in the sample (p<0.05; Z<Z0.05).
 Conclusions. TTV is not exclusively hepatotropic, but a multitropic opportunistic virus that is reactivated in an immunosuppressed organism, including primary minor immunodeficiencies with damage to various branches of the immune system. Artesunate, given a three-month course of 100-150 mg/day orally, provides the elimination of viral DNA from blood cells in 62% of cases with satisfactory tolerability, so it can be considered as a new promising drug for the treatment of this infection.