Blood Lymphocytes Of Patients 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.

Redox status of blood lymphocytes in patients with soft tissue sarcomas

Introduction. The proven significance of free radical disorders in oncopathology determines the relevance of their study in soft tissue sarcomas (STS), which are characterized by a tendency to recurrence and high mortality of patients.Aim. To study the indicators of free radical oxidation and antioxidant protection in blood lymphocytes in primary and recurrent STS, depending on the sex and age of patients.Materials and methods. In 51 patients with primary STS, T2bN0M0 (17 men and 34 women) and in 34 patients (14 men and 20 women) with relapses, the level of lipid peroxidation products – malondialdehyde and diene conjugates, activities of superoxide dismutase and glutathione peroxidase, total peroxidase activity, the level of reduced glutathione were studied by spectrophotometric methods. Patients were divided by age: ≤45 years and >45 years. The comparison groups (donors) consisted of 12 men and 17 women divided into the same age subgroups.Results. In men >45 years old with primary sarcomas, there was an increase in diene conjugates by 2.2 times, with relapses by 2.4 times, and in young men - only with relapses by 2.6 times. The level of malondialdehyde in men did not change with primary sarcomas, with relapses it was reduced in both age groups by 2.4–2.6 times. In women with primary STS, there was an increase in most of the studied parameters in both age groups by 1.6–1.9 times. The increase in diene conjugates level and superoxide dismutase activity was the most pronounced (>3 times) in patients of the older age group. The increase in glutathione peroxidase activity in primary STS in young women was 2.3 times, and in the older age group in patients of both sexes it was 5 times. In half of the women with relapses developed no earlier than 3 years after treatment, a decrease in both lipid peroxidation products by 2.6–3.4 times was found, and in all women almost 4-fold activation of superoxide dismutase and 8-fold glutathione peroxidase with an increase in the level of reduced glutathione by 7 times took place, which was significantly higher than in men. For women of the older age group, a 3-fold increase in the ratio of superoxide dismutase/total peroxidase activity is characteristic for primary STS and almost 5-fold for recurrence.Conclusion. In primary STS, the activation of lipid peroxidation products and antioxidant enzymes in women is more pronounced than in men. Changes in most indicators are more typical for patients of older age groups, in which relapse was accompanied by a decrease in malondialdehyde in patients of both sexes and maximum activation of superoxide dismutase and glutathione peroxidase in women.

Correction of immune status from hospitalized COVID-19-patients with immunotropic drug added to the basic treatment

Cellular immunity plays an important role in the control of SARS-CoV-2. Lymphopenia and a decrease in the functional activity of cells may be among the main reasons for deterioration of clinical outcomes of the disease. Usage of the bacterial therapeutic vaccine Immunovac-VP-4 during the inflammation phase may be promising for immunomodulation of the cellular immunity. The aim of our study was to evaluate the dynamics of lymphocyte subpopulations in hospitalized patients with COVID-19 upon combining the basic therapy with immunotropic drug based on the antigens from opportunistic pathogens. The study included 45 patients (18-70 years old) admitted with a confirmed diagnosis of moderate/severe infection caused by the COVID-19 virus. In addition to basic therapy, 33 persons of this group received Immunovac-VP-4 by a combined nasal-oral method. Subpopulation activity of peripheral blood lymphocytes in patients over time (at baseline, on the 14th and 30th day after hospitalization) was studied by flow cytometry by means of FC-500 Cytomics (Beckman Coulter, USA) using monoclonal antibodies (mAb) (Immunotech, France). In the group receiving only standard therapy, an increased number of T lymphocytes was detected on day 14 (79.9 (75.5-81.6), p = 0.00252), on day 30 from the start of treatment (78.4 (74.25-79. 2), p = 0.03662), and a decrease in B lymphocytes on day 14 (10.6 (7.78-11.63), p = 0.03236), on day 30 (7.85 (6.25-11.1), p = 0.01352) relative to baseline parameters upon admission. We revealed more pronounced changes in the parameters of cellular immunity relative to the initial parameters, i.e., an increased proportion of T lymphocytes on the 14th day (80.1 (73.8-84.2), p = 0.00018), and 30th day from starting the treatment (80.2 (76-81.9)), T helpers at 14 days after treatment (50.2 (43-57), p = 0.00694), cytotoxic T cells by 30th day of therapy (26.35 (24-29.4), p = 0.0114), decrease in B lymphocytes on day 14 (13.1 (8.2-16.9), p = 0 00158), on the 30th day from the start of treatment (8.2 (7.6-9.7), p <0.00001), and a transient decrease in NK cells on the 14th day (3.7 (2,1-6.3), p = 0.00308), with their recovery on the 30th day of observation to 8.6 (6-12.5) in the Immunovac-VP-4 group. Modulation of cellular immunity may be important for the virus clearance.

Bloom Syndrome Patients Display Accelerated Epigenetic Aging in Blood Lymphocytes

Bloom Syndrome (BSyn) is an autosomal recessive disorder caused by variants in the BLM genes at locus 15q26.1. The BLM gene encodes a protein that is part of the DNA RECQ helicase family, which has a role in homologous recombination, DNA structural integrity, DNA repair responses, and telomere maintenance. Patients with BSyn present with poor growth, sun sensitivity, mild immunodeficiency, diabetes, and increased risk of cancer, most commonly leukemias. Additionally, patients with BSyn develop myelodysplasia and life-threatening side effects from standard chemotherapy and radiation, often due to bone marrow toxicity. Cells from patients with BSyn display increased sister chromatid exchange, telomere defects, and telomere-dependent chromosome fusions. Despite these aging-associated findings, patients with BSyn do not appear to have typical premature aging characteristics such as graying hair, osteoporosis, and early-onset cardiovascular disease. We hypothesized that patients with BSyn may have increased epigenetic aging in blood, which may be related to their increased risk of hematologic dysfunction and malignancies. In this study, we aimed to use DNA methylation in order to identify an aging signature that could be a potential biomarker of disease risk. In partnership with the Bloom Syndrome Registry (BSR) as well as JScreen and our own clinical practice, we extracted 49 DNA samples from blood lymphocytes of patients with BSyn as well as carriers. The samples were then processed per standard Illumina protocol for the Infinium MethylationEPIC BeadChip kit. We utilized the online DNA Methylation Age Calculator (https://dnamage.genetics.ucla.edu/) to calculate multiple measures of methylation aging for each patient, including the Horvath pan-tissue epigenetic clock DNAmAge, the Hannum clock, the GrimAge lifespan predictor, and the PhenoAge phenotypic age estimate. Simple linear regression was used to determine statistical significance. Of the 49 total samples, 18 samples were from patients with BSyn (ages 1-38 years) and 31 samples were from carriers with one variant in BLM (ages 23-69 years). We discovered that BSyn patients had increased DNAm age compared to carriers measured by the pan-tissue clock (p=0.0054). Similarly, epigenetic clocks that were designed to predict human mortality risk (GrimAge and PhenoAge) indicate significant age acceleration as well, p<0.0088 and p<0.0001 respectively. Finally, DNA methylation-based estimators of telomere length show significantly shorter telomere lengths in BSyn patients compared to carriers, p<0.0001. Epigenetic age can be a better predictor of health and disease over biologic or chronologic age. In this study, we show for the first time that patients with BSyn have evidence of epigenetic aging across several measures in blood lymphocytes. Future studies are ongoing to investigate underlying mechanisms for this and whether epigenetic aging in these patients can predict hematologic dysfunction and cancer risk.

Lymphocyte Transformation Test Based on Lymphocyte Changes Observed by a Hematology Analyzer before and after Phytohemagglutinin Stimulation.

The lymphocyte transformation test is a classical test for the detection of cellular immune function and is based on subjective judgment. In this study, we have established an objective novel lymphocyte transformation test using the hematology analyzer to observe lymphocyte transformation. Whole blood cells were cultured using a whole blood method with a lymphocyte culture medium; phytohemagglutinin was used to stimulate the experimental samples, and control was set up at the same time. After the whole blood cells were cultured, the number of lymphocytes in the two groups was observed using a hematology analyzer, and the conversion rate was calculated. The new method was used to observe differences in lymphocyte conversion in the peripheral blood of patients with hematopathy and healthy persons. There were significant differences between the stimulated peripheral blood group and the blank group. The transformation rate of peripheral blood lymphocytes in patients with hematopathy was significantly lower than that in healthy persons; the difference was statistically significant (P < 0.05). Lymphocyte transformation can be observed using a hematology analyzer. The lymphocyte transformation test that is based on the determination of lymphocyte count by a hematology analyzer has important clinical value.

Analysis of annexin V levels and cytokine status in patients with acute myocardial infarction

Aim. Study of annexin V levels and cytokine status in patients with acute myocardial infarction. Materials and methods. We examined 27 patients with Q-forming myocardial infarction who were admitted 12–24 hours after the development of the clinic, and 20 individuals in the control group. The lipid spectrum was determined using an automatic analyzer Humalizer-3000 (Human, Germany). Immunological studies were performed on a flow cytofluorimeter Navios (Beckman Coulter, USA). Lymphocyte subpopulations were evaluated using monoclonal antibodies produced by Immunotech (Coulter Corporation, USA). A set of ANNEXIN V-FITC/7 AAD (Beckman Coulter, USA) was used to isolate cells that had undergone apoptosis. Isolation of peripheral blood mononuclears was performed by centrifugation on a density gradient Ficol-hypac (Pharmacia Fine Chemicals, Sweden), interleukin-1β was determined using the Vector-Best test system (Vector-Best, Russia), and the tumor necrosis factor was determined using the RayBio Human TNF-alpha ELISA Kit (USA). Statistical data processing was performed using the program package Statistica 10.0. Results. In patients with acute myocardial infarction on the background of dyslipidemia (total cholesterol and LDL cholesterol increased, cholesterol-HDL reduced) showed a significant decrease in the total number of T-lymphocytes (СD 3+) (p &lt; 0.05), T-suppressor (СD 8+) (p &lt; 0.01), B-cells number (СD 19+) and the ratio of CD 4+/CD 8+ lymphocytes increased (p &lt; 0.05). In patients with myocardial infarction there was a significant increase of apoptotic cells and рro-inflammatory cytokines involved in apoptosis (IL-1β and TNF-α) in the blood compared with the control. Conclusion. Increased spontaneous apoptosis of peripheral blood lymphocytes in patients with acute myocardial infarction indicates the systemic nature of apoptosis in acute coronary pathology, due to increased annexin-dependent apoptosis of T-lymphocytes as a result of immune disorders, which leads to activation of the cytokine system with an increased concentration of рro-inflammatory mediators (TNF-α, IL-1β). The identified dependencies require further study with the prospect of developing predictive algorithms for myocardial infarction outcomes based on indicators of annexin V and pro-inflammatory cytokines.

Diagnostic Value of Peripheral Blood Lymphocytes for Primary Immune Thrombocytopenia.

The purpose of this study was to investigate the absolute value of peripheral blood lymphocytes in patients with primary immune thrombocytopenia and the diagnostic effect on patients with primary immune thrombocytopenia. From January 2020 to June 2021, 76 patients with primary immune thrombocytopenia and 80 healthy check-ups admitted to our hospital were selected as study subjects and divided into a control group (80 patients, healthy check-ups) and an observation group (76 patients, primary immune thrombocytopenia), according to the health status of the organism. Early morning fasting venous blood was collected from both groups, and the absolute value of peripheral blood lymphocytes was measured and compared using a fully automated hematology analyzer to investigate the diagnostic value of absolute peripheral blood lymphocytes in primary immune thrombocytopenia. The CD3+, CD3+CD4+, CD4+/CD8+, and CD16+CD56+ assay values in the observation group were lower than those in the control group, and the CD3+CD8+, CD19+, and ALC assay values were higher than those in the control group (P < 0.05). The CD3+CD8+ detection values of newly diagnosed patients were similar to those of relapsed refractory patients (P > 0.05); CD3+, CD3+CD4+, CD4+/CD8+, and CD16+CD56+ detection values of newly diagnosed patients were lower than those of relapsed refractory patients, and CD19+ and ALC detection values were higher than those of relapsed refractory patients; CD3+, CD3+CD4+, CD4+, CD4+/CD8+, and CD16+CD56+ detection values of mild patients were lower than those of relapsed refractory patients; CD3+, CD3+CD4+, CD4+/CD8+, and CD16+CD56+ detection values were higher in mild patients than in severe patients, and CD3+CD8+, CD19+, and ALC detection values were lower than in severe patients (P < 0.05). The absolute lymphocyte values were of high diagnostic value in primary immune thrombocytopenia, with a sensitivity and specificity of 93.42% and 90.00%. The application of absolute peripheral blood lymphocyte value in the clinical diagnosis of primary immune thrombocytopenia can achieve a better detection and diagnosis effect, which has a positive impact on the early diagnosis rate and can help patients to obtain more timely, effective and targeted treatment, and is worthy of promotion.

Fluorescent In Situ Staining and Flow Cytometric Procedures as New Pre-Diagnostic Tests for Sialidosis, GM1 Gangliosidosis and Niemann-Pick Type C.

Background: Early diagnosis is essential in the field of lysosomal storage disorders for the proper management of patients and for starting therapies before irreversible damage occurs, particularly in neurodegenerative conditions. Currently, specific biomarkers for the diagnosis of lysosomal storage disorders are lacking in routine laboratory practice, except for enzymatic tests, which are available only in specialized metabolic centers. Recently, we established a method for measuring and verifying changes in GM1 ganglioside levels in peripheral blood lymphocytes in patients with GM1 gangliosidosis. However, fresh blood is not always available, and using frozen/thawed lymphocytes can lead to inaccurate results. Methods: We used frozen/thawed fibroblasts obtained from stored biopsies to explore the feasibility of fluorescent imaging and flow-cytometric methods to track changes in storage materials in fibroblasts from patients with three lysosomal neurodegenerative conditions: GM1 gangliosidosis, Sialidosis, and Niemann–Pick type C. We used specific markers for each pathology. Results and Conclusions: We demonstrated that with our methods, it is possible to clearly distinguish the levels of accumulated metabolites in fibroblasts from affected and unaffected patients for all the three pathologies considered. Our methods proved to be rapid, sensitive, unbiased, and potentially applicable to other LSDs.

Analysis of Cytokine Production Profiles of Local and Systemic Lymphocytes in Sick Building Syndrome Compared with Ocular Allergy.

PurposeWe have previously studied clinical and allergological aspects of sick building syndrome (SBS) cases with ocular disorders and found that SBS is suggested to be partially induced by an allergic response. We analyzed the cytokine production profiles of conjunctival and peripheral blood lymphocytes in patients with SBS with ocular manifestations to further evaluate the pathophysiology of SBS from an immunological standpoint.MethodsWe obtained conjunctival samples and peripheral blood mononuclear cells (PBMC) from 15 cases of SBS with ocular findings, 49 cases of allergic conjunctival diseases (ACD) (allergic conjunctivitis (AC), atopic keratoconjunctivitis (AKC), and vernal keratoconjunctivitis (VKC)), and normal controls. Frequencies of cytokine-producing T cells were analyzed by flow cytometry based on an intracellular cytokine staining method.ResultsAlthough no significant difference was observed in the percentage of interferon (IFN)-γ-producing CD4+ T cells in PBMC between patients with SBS and controls, the percentage of interleukin (IL)-4-producing PBMC CD4+ T cells in patients with SBS was significantly higher than that in controls. The percentage of IL-4-producing CD4+ T cells in the conjunctiva in patients with SBS was significantly higher than that in controls, whereas it was significantly lower than that in AKC and VKC. A significant correlation was observed between the percentage of IL-4-producing CD4+ T cells in the conjunctiva and clinical score.ConclusionThese results suggest that SBS may be a kind of allergic disorder and that IL-4 plays a role in the development of allergic disorders in SBS ocular lesions.

Оценка факторов риска в течении ревматоидного артрита и анкилозирующего спондилита

The purpose — to study individual risk factors and their influence on the course of rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Material and methods. Patients with RA and AS took part in the research; the group of comparison was practically healthy people. We evaluated clinical condition and laboratory indicators (the level of cytokines, lymphocyte subpopulation, rheumatoid factor, antibodies to the cyclic citrullinated peptide, S-reactive protein, proinflammatory cytokines, monocyte-macrophage protein, vascular endothelial factor, and cell cycle analysis). The obtained data were processed with Statistics 7.0 software. The results were considered to be statistically reliable at р &lt; 0.05. Results. Hyper production of proinflammatory cytokines was marked in sick patients compared with healthy ones. The analysis of peripheral blood lymphocytes in patients showed their excess in comparison to healthy people. The assessment of lymphocytes ability for blast transformation defined the lymphocytes activation with the growth of cells at apoptosis stage (G1-phase); with the decrease in the proportion of resting cells (GO-phase) in patients, the activation coefficient was significantly growing. More apoptosis activation markers were found in the group of RA patients. Changes in apoptosis were revealed with the decreased proportion of lymphocytes in unstimulated cultures of patients; at the same time, the amount of cells in apoptosis increased four times in the examined patients compared to the healthy ones. Conclusions. In patients with RA and AS, changes of the factors of the immune system wee noted as the amount of pro-inflammatory cytokines IL—1β, IL-6, TNF—α and the concentration of MSR-1, VEGF, SPB, RF increased. The enhancement of cellular apoptosis is due to the increase of the apoptosis inducer — CD95+9(FaS) lymphocytes, TNF- α, the growth of cells at pre-synthetic stage of apoptosis G1-phase and the activation coefficient.

Analysis of Pathogenic Bacteria of Mooren’s Ulcer and T Lymphocyte Activation

To analyze the distribution and types of pathogenic bacteria of Mooren’s ulcer and the activation mechanism of T lymphocytes to provide reference for the treatment of Mooren’s ulcer, 156 patients (162 eyes) who were in the hospital were rolled into the observation group. During the same period, 134 healthy people were rolled into the control group. The distribution of infectious pathogens in the observation group was identified. Then, flow cytometry was adopted to separate and detect the peripheral blood lymphocytes of patients, and RT-PCR was used to detect levels of the transcription factor T-bet, GATA-3, and Stat5 in peripheral blood mononuclear cells (PBMCs). It was found that fungal pathogens accounted for 43.59%; the bacterial infection rate was 40.38%. In the observation group, the CD4, CD8, and C25 were expressed more (P &lt; 0.01), and the CD45 and CD45R were expressed less than the control group (P &lt; 0.05); the proportion of Th1 cells was obviously higher (P &lt; 0.01); the expression of T-bet and GATA-3 was obviously higher (P &lt; 0.05), the percentage of HLA-DR in CD4+ and HLA-DR, CD-25, and CD69 in CD8+ positive cells was obviously higher (P &lt; 0.05). In conclusion, fungal infection rate of Mooren’s ulcer is relatively high, peripheral blood T cells and their subgroups are abnormally activated, and T cell activation is related to the pathogenesis of Mooren’s ulcer.

Cytokine production by peripheral blood lymphocytes in patients with ovarian epithelial tumors

In presence of autoserum, production of IL1b, IL2, IL4, IL6 and TNFa by peripheral blood lymphocytes of 30 patients with epithelial tumors of ovaries and 50 patients with epithelial ovarian cancer of I-IV stages was investigated. The data received enable to ascertain the increase of mononuclear activity during ovarian tumor progression, increase of T-helpers of the second type activity, along with the reduction of T-helpers of the first type activity in peripheral blood of patients, and abnormalities of IL6-dependent mechanism of control of ILIb and TNFa production by peripheral blood lymphocytes of patients with ovarian cancer.

Зміни гліканових детермінант мембран лімфоцитів периферійної крові хворих на B–клітинний хронічний лімфолейкоз за умов протипухлинної терапії

The purpose of the study was to study the nature of changes in the exposure of surface glycans of peripheral blood lymphocytes in patients with B-cell chronic lymphocytic leukemia under conditions of antitumor therapy. Materials and methods. We studied the features of exposure of surface glycotopes of peripheral blood lymphocytes in patients with B-cell chronic lymphocytic leukemia under conditions of antitumor therapy using a set of seven lectins labeled with FITC and monoclonal antibodies to Tn-antigen- FITC for the detection of Tn antigen and CD43 exposure on blood lymphocytes. Cytostatic therapy included cyclophosphamide, vincristine (oncovin), prednisolone. Data were recorded on a Beckman Coulter EPICS flow cytometer. The results were processed using FCS3 Express. Results and discussion. The number of lymphocytes of healthy donors with a positive reaction to ConA, PHA-L, SNA, MAA-II and α1-acid glycoprotein amounted to 16.0±3.0%, 23.0±2.3%, 15.0±1.5%, 25.0±1.8% and 15.0±1.3%, respectively. The number of LABA-, UEA I-positive lymphocytes was 0.90±0.03% and 2.9±0.2%, respectively, and there was no binding to antibodies to Tn- and CD43-antigens. In the blood of patients with chronic lymphocytic leukemia, the level of ConA-, SNA- and MAA-II-positive lymphocytes increased relative to control by 2.2, 3.7 and 2.6 times, respectively. The number of LABA- and UEA I-positive lymphocytes in patients with chronic lymphocytic leukemia increased by 11 (p &lt;0.01) and 23 (p &lt;0.001) times and amounted to 10.5±0.5% and 67.5±5.5% respectively. The number of lymphocytes with CD43 antigen on their surface increased by 72 times, and the Tn antigen increased by 80 times. Cytostatic therapy reduced the level of LABA- and UEA I-positive lymphocytes by almost half, and MAA II-positive cells and lymphocytes interacting with antibodies to CD43 and Tn antigen by a third. The level of PHA-L-positive lymphocytes in the blood of chronic lymphocytic leukemia patients after undergoing alkylating therapy increased by 18.0±2.0% and almost did not differ from those obtained in the control group. Conclusion. 1. In chronic lymphocytic leukemia patients, the structure of glycoconjugates in peripheral blood lymphocytes changes, manifested in increased exposure of L-fucose, α-mannose and N-acetylneuraminic acid, which is confirmed by a significant increase in relation to the control of the number of ConA-, SNA-, MAA-II-, LABA I-positive cells. 2. Patients with chronic lymphocytic leukemia showed a significant increase in the number of lymphocytes, in which the markers of carcinogenesis CD43 and Tn antigens were found. 3. Cytostatic therapy significantly reduced the level of LABA-, UEA I- and MAA II-positive cells, as well as partially Tn- and CD43-antigen-positive lymphocytes, which indicates its positive effect on the treatment of chronic lymphocytic leukemia

Дослідження глікобіому поверхні лімфоцитів хворих на В-клітинний хронічний лімфолейкоз

The purpose of the study was to investigate the intensity of exposure of peripheral blood lymphocyte surface glycans in patients with B-cell chronic lymphocytic leukemia by measuring the density of lectin- or antigen-positive epitopes under antitumor therapy in order to evaluate it for a more reasonable selection of qualitative and quantitative composition of therapy. Materials and methods. The objects of the study were blood lymphocytes of patients with chronic lymphocytic leukemia (n=15) aged 58-66 years before and after a course of standard chemotherapy according to the COP scheme. The control group consisted of healthy volunteers (n=15) aged 55 to 65 years. Isolation of lymphocytes was performed by a modified method of A. Boyum. Polyclonal antibodies to α1-acid glycoprotein and fibronectin were used. Exposure to Tn antigen and CD43 on blood lymphocytes was determined with secondary antibodies to mouse immunoglobulins conjugated to FITC (Millipore, USA). To study the exposure of glycans on the surface of lymphocytes, we used a set of seven lectins labeled with FITC. Data recording was performed on a Beckman Flower EPICS flow cytometer. Processing of the results was done using the program FCS3 Express. Results and discussion. Compared with the group of hematologically healthy donors on the surface of lymphocytes in patients with chronic lymphocytic leukemia, a 20-fold increase in the density of exposure to ConA epitopes, 10 times – UEAI- and SNA-positive epitopes were shown; MAA II epitope, Tn, and CD43 antigen densities were increased 100-fold (p &lt;0.01). Exposure densities of MAA II-, Tn-, and CD43-positive epitopes on the plasma membrane of lymphocytes in patients with chronic lymphocytic leukemia receiving alkylation therapy decreased 10-fold relative to treatment data, but remained 10-fold higher than in the group of healthy hematologists. Conclusion. On the plasma membranes of lymphocytes in patients with chronic lymphocytic leukemia, the density of exposure of mannose and neuraminic acid residues was significantly increased. COP therapy reduced the density of these epitopes to control values. A significant increase in the density of carcinogenesis markers – Tn- and CD43-antigens on the plasma membranes of lymphocytes in patients with chronic lymphocytic leukemia has been shown. COP therapy provided only a partial decrease in their density, which indicates the insufficient effectiveness of COP therapy, its inability to completely stop the oncological process in patients with chronic lymphocytic leukemia

Structural and functional state of blood lymphocytes in patients with infectious mononucleosis with different course

The article presents the results of our own studies. The aim was to determine the structural and functional status of blood lymphocytes in patients with acute and prolonged course of infectious mononucleosis (IM) in children. Materials and methods. 102 children were under clinical and laboratory-instrumental supervision, the children were divided into groups: group 1 – 65 children with IM with an acute course of the disease; group 2 – 37 children with a prolonged course of the disease. All children underwent standard clinical laboratory and instrumental laboratory examinations. The diagnosis of IM was confirmed by PCR (detection of EBV DNA in the blood) and ELISA (anti-EBV IgM and IgG). Research results. In the study of the structural state of the cytoplasmic membrane of the lymphocytes in the blood of patients with MI in the onset of the disease, it was found that the average values of penetration rate of the electron paramagnetic resonance of spin probes (PR EPR s. p.) in children of both groups were significantly higher than normal (P &lt; 0.001). There are also differences between groups of patients. In this case, the value of PR EPR s. p. in patients with a prolonged course by 15.8 % exceeded those in patients with acute IM (P &lt; 0.001). According to the rate of microviscosity of the intracellular content (MV IC), its values were reduced compared with the control – by 22.1 % (P &lt; 0.001) in patients with acute course of the disease and by 25.1 % – with a prolonged course of IM). In addition, in patients with a prolonged course of the disease, the values were 9 % lower than in the group with acute infectious mononucleosis. When considering immunological parameters, it was found that the indicators of the T-immune system for patients with a prolonged course of the disease in comparison with the alternative group was characterized by a decrease in the content of CD3 &lt;50 % (respectively in 51.3 % and 26.2 % of patients; P &lt; 0.05); CD4 &lt;31 % (62.1 % and 32.4 %, respectively; P &lt; 0.05) and CD8 &lt;15 % (37.8 % and 10.8 %, respectively; P &lt; 0.01). With regard to the cytokine profile, the level of IL-1 &lt;20.0 pg/ml was determined 3.5 times more often in patients with a prolonged course of the disease compared to the acute course (64.8 % and 18.5 % of patients, respectively); TNFα &lt;20.0 pg/ml 1.9 times more often (48.6 % and 24.6 %, respectively) and a very high (&gt;30.1 pg/ml) level of IL4 in 40.5 % and 20 %). From the B-system of immunity in patients with a prolonged course of IM in comparison with the acute course increased content of CD22 was more often determined, as well as low levels of IgA, IgM &lt;1.1 g/l and IgG &lt;10.0 g/l. Conclusions. According to the results of observations, the pathogenetic role of the violation of the structural organization of blood lymphocytes in the formation of IM is established. It was found that these disorders in the form of increased permeability of their cytoplasmic membrane and reduced viscoelastic properties of their intracellular environment are more pronounced with a prolonged course of the disease, which is a factor in the prolongation of the disease. It is determined that the indicators of cellular and humoral parts of the immune system affect the course of IM. During formation of an acute course of IM in children already in the acute period of a disease activation of both cellular and humoral links of immunity, which is shown in the form of increase in relative content of CD3+, CD4+, CD8+ and CD22+ and levels of immunoglobulins M, A, is noted. For the prolonged course of the disease depression of T-cell immunity in the form of a decrease in the relative content of CD3+, CD4+ and CD8+ lymphocytes and an increase in CD22+, as well as inhibition of antibody genesis are characteristic. It was found that the variant of IM depends on the type of reaction of T-helper clones, namely – in the initial period of manifestation of IM with its acute course there is activation of T1 and T2 helper response, which manifests itself in a significant increase in IL-1, TNFα and moderate IL-4. Prolonged course of the disease is formed against the background of weak activation of pro-inflammatory interleukins (IL-1, TNFα) and significant – anti-inflammatory IL-4.

Diagnostic Value of 64 Slice Spiral CT Combined Detection of Cd4 + Cd28-T Lymphocytes in Patients with Coronary Artery Plaque

in recent years, with the continuous progress and development of science and technology and the increasingly mature medical technology, the diagnosis of coronary artery plaque is becoming more and more complex. In recent years, there are many challenges and obstacles in the development of scientific and medical treatment. In order to successfully break through these challenges and obstacles, we must establish a 64- layer spiral CT detection system and lymphocyte symptom response. This paper mainly introduces the design and study of the diagnostic value of 64 slice spiral CT combined detection of CD4 + CD28-T lymphocytes in peripheral blood of patients with coronary plaque. This paper uses 64 slice spiral CT to detect the value of peripheral blood CD4 + CD28-T lymphocytes in the diagnosis of coronary plaque. The main purpose of CT is to solve two problems: the shape, size, location and activity of coronary plaque; The influence of coronary plaque on vascular anatomy and physiology plays a guiding role in treatment. The motion of coronary plaque and its relationship with heart cavity, heart wall and valve mouth (such as whether the movement of coronary plaque causes obstruction of valve mouth, etc.) can be displayed by film scanning, and cardiac function can be calculated. The results show that the diagnostic value of combined detection of CD4 + CD28-T lymphocytes in peripheral blood of patients with 64 slice spiral CT makes the plaque treatment rate increase by 16%. The limitations of the design of 64 slice spiral CT are analyzed the research and summary will enrich the academic research results.

Comparative analysis of physical doses and biomarker changes in subjects underwent Computed Tomography, Positron Emission Tomography-Computed Tomography, and interventional procedures

Even though the medical uses of ionizing radiation are well-acknowledged globally as vital tools for the improvement of human health, they also symbolize the major man-made sources of radiation exposure to the population. Estimation of absorbed dose and biological changes after radiation-based imaging might help to better understand the effects of low dose radiation. Because of this, we measured the Entrance Surface Dose (ESD) at different anatomical locations using Lithium tetraborate doped with manganese (Li2B4O7: Mn), recorded Dose Length Product (DLP) and Dose Area Product (DAP), analyzed Chromosomal Aberration (CA), Micronucleus (MN), gamma-H2AX (γ-H2AX), and p53ser15 proteins in the blood lymphocytes of patients (n = 267) underwent Computed Tomography (CT), Positron Emission Tomography-CT (PET/CT), and interventional procedures and healthy volunteers (n = 19). The DLP and effective doses obtained from PET/CT procedures were significantly higher (p < 0.05) when compared to CT. Fluoroscopic time and DAP were significantly higher (p < 0.05) in therapeutic compared to diagnostic interventional procedures. All the anatomical locations registered a significant amount of ESD, the ESD obtained from CT and interventional procedures were significantly (p < 0.05) higher when compared to PET/CT. Fluoroscopic time did not correlate with the ESD (eye, head, thyroid, and shoulder; R2 = 0.03). CA frequency after PET/CT was significantly higher (p < 0.001) when compared to CT and interventional procedures. MN frequency was significantly higher in 24-hs (p < 0.001) post-interventional procedure compared to 2-hs. The mean ± SD of mean fluorescence intensity of γ-H2AX and p53ser15 obtained from all subjects underwent PET/CT and interventional procedures did not show a significant difference (p > 0.05) between pre- and post-procedure. However, the relative fluorescence intensity of γ-H2AX and p53ser15 was >1 in 58.5 % and 65.8 % of subjects respectively. Large inter-individual variation and lack of correlation between physical dose and biomarkers suggest the need for robust dosimetry with a large sample size to understand the health effects of low dose radiation.

Апоптоз лімфоцитів крові у хворих на автоімунний тиреоїдит і шляхи корекції

Мета — дослідити деякі ланки патогенезу розвитку вузлового зоба на тлі автоімунного тиреоїдиту (АІТ) шляхом вивчення активності процесів пероксидного окиснення ліпідів і білків, апоптозу лімфоцитів периферичної крові та рівня деяких цитокінів до та після оперативного втручання, розробити ефективні методи їх корекції. Матеріали та методи. Обстежено 80 жінок із вузловим зобом на тлі АІТ. Результати. Показано, що порушення програмованої загибелі лімфоцитів при АІТ проявляється збільшенням вмісту фактора некрозу пухлини α і CD95+ лімфоцитів на тлі зниження числа апоптичних клітин. Висновки. Після проведеного комплексного лікування відзначена позитивна динаміка порушених показників імунного статусу, насамперед нормалізація порушеного балансу субпопуляції апоптичних Т-лімфоцитів та антитіл до тиреоглобуліну та тиреоїдної пероксидази, зниження вмісту цитокінів і повернення в межі нормальних значень рівнів гормонів щитоподібної залози.

Study of genes involved in angiogenesis and metabolic processes of peripheral blood lymphocytes in patients with chronic endometritis

Objective. To study the polymorphisms of the genes involved in angiogenesis and in metabolic processes, to assess the level of lymphocytes in patients with chronic endometritis and practically healthy women of reproductive period.&#x0D; Materials and methods. 86 patients were examined; DNA regions of the genes eNOS 1799983 (Glu298Asp), PPARA (G2528C), ApoE rs429358 (Cys130Arg), MTHFR (C677T, A1298C) were used as primers; blood lymphocytes (CD3+, CD4+, CD8+, CD19+, CD95+) were assessed.&#x0D; Results. Statistically significant differences in gynecological and chronic somatic pathology were obtained in patients with chronic endometritis; they more often than practically healthy women had polymorphisms of the genes ApoE rs429358, eNOS1799983, PPARA (G2528C); patients with chronic endometritis more often had dysregulation of the immune system in the form of insufficiency of the cellular effector link of immunity and changes in the PPARA, ApoE, eNOS gene. Attention was drawn to the obtained relationships of polymorphic genes and clinical manifestations in patients with chronic endometritis, in particular, with a history of non-developing pregnancy in anamnesis, there was more often detected polymorphism of the ApoE gene, with abnormal uterine bleeding polymorphism of PPARA, with chronic inflammatory pathology of the gallbladder polymorphism of the MTHFR gene.&#x0D; Conclusions. The prevalence of polymorphism of the genes eNOS 1799983 (Glu298Asp), PPARA (G2528C), ApoE rs429358 (Cys130Arg), MTHFR (C677T, A1298C) was obtained in patients with chronic endometrial inflammation compared with practically healthy participants in the study. Insufficiency of the cellular effector link of immunity was revealed in the majority of patients with ChE and an association with allele C genotypes G/C and C/C of PPARA 4253778 gene, with allele C genotypes G/C and C/C of ApoE42935 gene, with allele C genotypes G/C and C/C of eNOS 1799983 gene and G/C genotype of MTHFR gene (C677T, A1298C).

PERIPHERAL BLOOD MONONUCLEAR DNA DAMAGE IDENTIFIED BY THE «СOMET» METHOD, AS A POSSIBLE INDICATOR OF SENSITIVITY OF MELANOMA TO IMMUNOTHERAPY WITH NIVOLUMAB

P urpose of the study : to assess whether the determination of the grade of dNA damage in the blood lymphocytes of patients with metastatic melanoma can be a criterion for assessing the effectiveness of therapy. Material and Methods. The grade of dNA damage in blood mononuclear cells was studied using the comet assay in 10 patients with progressive metastatic melanoma before therapy with Nivolumab and 3–4 months after Nivolumab therapy. Results. It was revealed that prior to treatment the level of dNA damage in blood mononuclears was higher in patients with melanoma than in controls (healthy subjects). This level significantly decreased when stable disease or complete regression were reached, while it changed less significantly or increased in cases with disease progression. Сonclusion. The initial level and changes in lymphocyte dNA damage in patients with melanoma can serve as a criterion for assessing tumor response to immunotherapy with Nivolumab.