Peripheral Blood Lymphocytes Of Patients Research Articles

Relationship between onco-immunological and morphologic characteristics of lymphoepithelioma-like carcinoma and lymphocyte subtypes of peripheral blood

Objective: To study the relationship between the onco-immunological and morphologic characteristics of lymphoepithelioma-like carcinoma (LELC) and peripheral blood lymphocyte subtypes and its clinical significance. Methods: The pathologic and clinical data of 117 LELC patients who were admitted to the Tumor Hospital of the University of Chinese Academy of Sciences from 2006 to 2018 were collected. The histological classification was based on previously reported morphological classification method. The onco-immunological and morphologic characteristics of the tumors such as lymphoid follicle formation and interstitial fibrous hyperplasia, patient's peripheral blood lymphocyte subtypes and prognosis data were collected. The relationship between various factors and their impact on prognosis were analyzed. Results: There were 117 patients, including 61 females and 56 males. The male to female ratio was 0.9∶1.0. The age of onset was 24-89 years (median 52 years). Primary sites included head and neck (68 cases), lungs (26 cases), stomach (15 cases), and others (eight cases). Morphologically, 54 cases were type Ⅰ, 62 cases were type Ⅱ, and one case could not be classified. The onco-immunological and morphologic features of the LELC tumors showed a continuous spectrum. Interstitial TILs were noted from focally to diffuse, and the interstitial fibrous tissues were from hardly visible to obvious sclerotic. Formation of lymphoid follicles was seen in 42 patients; obvious fibrosis was seen in 31 cases. Data of peripheral blood lymphocyte subtyping by flow cytometry were available in 73 cases. These data included CD3+total T cells, CD3+CD4+helper T cells, CD3+CD8+cytotoxic T cells, CD3-CD56+natural killer (NK) cells, CD3-CD19+B cells, CD4+CD45RA-T helper induction subgroup, CD4+CD45RA+ T suppression induction subgroup, CD4+CD45RO+memory T cell subgroup, CD45RA+CD45RO+activated T cell subgroup, CD8+CD38+activated cytotoxic T cell, and CD25+lymphocytes and CD44+lymphocyte. The proportion of lymphocytes of each subtype was normal in most patients, but the proportion of CD44+lymphocytes in 61 cases (83.6%) was increased; the proportion of T cell suppression induced subgroups was decreased in 53 cases (72.6%). Correlation analysis found a significant correlation between clinical stage and NK cells (P=0.023); tumor histologic type and cytotoxic T cells were significantly positively correlated (P=0.012); while tumor cell morphologic differentiation was significantly related to total T cells (P=0.003) and NK cells (P=0.026); Formation of interstitial lymphoid follicles was positively correlated with memory T cell subsets (P=0.025); Tumor interstitial fibrosis was significantly positively correlated with T suppression-induced subpopulations (P=0.004), and was significantly negatively correlated with total T cells (P=0.023) and with the expression of CD44 adhesion molecules (P=0.003). Survival analysis found that lymphoid follicle formation was a favorable prognostic factor for LELC (P=0.001). Conclusions: The onco-immunological and morphologic features in LELC show a continuous spectrum; the tumor clinicopathological characteristics and onco-immunological morphology are closely related to peripheral blood T lymphocyte subtypes, and the formation of interstitial lymphoid follicles is a favorable prognostic factor for LELC.

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.

Serotherapy as Graft-Versus-Host Disease Prophylaxis in Haematopoietic Stem Cell Transplantation for Acute Lymphoblastic Leukaemia.

Serotherapy comprising agents such as anti-thymocyte globulin, anti-T-lymphocyte globulin, and the anti-CD52 monoclonal antibody alemtuzumab is used widely to reduce the incidence of graft-versus-host disease (GvHD) after paediatric haematopoietic stem cell transplantation (HSCT). The outcome of transplants using matched unrelated donors now approaches that of matched sibling donors. This is likely due to better disease control in recipients, the use of donors more closely human-leukocyte antigen (HLA)-matched to recipients, and more effective graft-versus-host disease (GvHD) prophylaxis. The price paid for reduced GvHD is slower immune reconstitution of T cells and thus more infections. This has led to studies looking to optimise the amount of serotherapy used. The balance between prevention of GvHD on one side and prevention of infections and relapse on the other side is quite delicate. Serotherapy is given with chemotherapy-/radiotherapy-based conditioning prior to HSCT. Due to their long half-lives, agents used for serotherapy may be detectable in patients well after graft infusion. This exposes the graft-infused T cells to a lympholytic effect, impacting T-cell recovery. As such, excessive serotherapy dosing may lead to no GvHD but a higher incidence of infections and relapse of leukaemia, while under-dosing may result in a higher chance of serious GvHD as immunity recovers more quickly. Individualised dosing is being developed through studies including retrospective analyses of serotherapy exposure, population pharmacokinetic modelling, therapeutic drug monitoring in certain centres, and the development of dosing models reliant on factors including the patient's peripheral blood lymphocyte count. Early results of “optimal” dosing strategies for serotherapy and conditioning chemotherapy show promise of improved overall survival.

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 < 0.01), and the CD45 and CD45R were expressed less than the control group (P < 0.05); the proportion of Th1 cells was obviously higher (P < 0.01); the expression of T-bet and GATA-3 was obviously higher (P < 0.05), the percentage of HLA-DR in CD4+ and HLA-DR, CD-25, and CD69 in CD8+ positive cells was obviously higher (P < 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.

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 < 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.

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.

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

Cisplatin Reduces the Frequencies of Radiotherapy-Induced Micronuclei in Peripheral Blood Lymphocytes of Patients with Gynaecological Cancer: Possible Implications for the Risk of Second Malignant Neoplasms.

Gynaecologic cancers are common among women and treatment includes surgery, radiotherapy or chemotherapy, where the last two methods induce DNA damage in non-targeted cells like peripheral blood lymphocytes (PBL). Damaged normal cells can transform leading to second malignant neoplasms (SMN) but the level of risk and impact of risk modifiers is not well defined. We investigated how radiotherapy alone or in combination with chemotherapy induce DNA damage in PBL of cervix and endometrial cancer patients during therapy. Blood samples were collected from nine endometrial cancer patients (treatment with radiotherapy + chemotherapy—RC) and nine cervical cancer patients (treatment with radiotherapy alone—R) before radiotherapy, 3 weeks after onset of radiotherapy and at the end of radiotherapy. Half of each blood sample was irradiated ex vivo with 2 Gy of gamma radiation in order to check how therapy influenced the sensitivity of PBL to radiation. Analysed endpoints were micronucleus (MN) frequencies, apoptosis frequencies and cell proliferation index. The results were characterised by strong individual variation, especially the MN frequencies and proliferation index. On average, despite higher total dose and larger fields, therapy alone induced the same level of MN in PBL of RC patients as compared to R. This result was accompanied by a higher level of apoptosis and stronger inhibition of cell proliferation in RC patients. The ex vivo dose induced fewer MN, more apoptosis and more strongly inhibited proliferation of PBL of RC as compared to R patients. These results are interpreted as evidence for a sensitizing effect of chemotherapy on radiation cytotoxicity. The possible implications for the risk of second malignant neoplasms are discussed.

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).

Characterization of Shifts of the Cellular Component the Immune Status of Patients with Long-Term Course of Chronic Post-Traumatic Uveitis

Purpose: to study the subpopulation composition of peripheral blood lymphocytes in patients with long — term chronic post-traumatic uveitis (CPT).Patients and Methods. There were 131 people aged 17 to 87 years, who received the surgical treatment in the Department of plastic surgery and eye prosthetics in NMITS eye diseases Helmholtz of the Ministry of health, Russia, where the removal of the eyeball by evisceration was carried out in 101 patients (70.3 %), 30 patients (29.7 % of cases) were enucleated. The study identified two groups: the Ist group– with the outcome of the injury, including non-penetrating (n = 46) and penetrating wounds (n = 51) of the eyes, were 97 patients; group II included 34 patients with complications due to single (n = 8) or repeated (n = 26) ophthalmic surgery (development of glaucoma and retinal detachment).Results. When analyzing the mean values, we found a decrease in the relative number of CD3+ CD4+ helpers/inducers in the blood of patients of both groups (up to 43.5 ± 0.81 and 42.19 ± 1.29 % versus 45.7 ± 1.19 % in the control), statistically confirmed for the 2nd group. Individual analysis revealed multidirectional shifts from the norm of cellular immune status of patients with PU in both groups. The most interesting, in our opinion, results were obtained by analyzing the relative number of effector cytotoxic cells of adaptive and nonspecific immunity. The increase in the relative content of t-cytotoxic lymphocytes CD3+ /CD8+ and NK-lymphocytes CD16+ /CD56+ was revealed with the same frequency, approximately in 18.5 and 19.5 % of cases in the PU group at the outcome of injury, in the 2nd group the increased number of these subpopulations was observed more often, amounting to 23.5 and 20.5 %, respectively.Conclusions. The results of the study on a large clinical material indicate that in long-term CPTU there is a pronounced systemic imbalance in the immunoregulatory subpopulations of lymphocytes, revealed by individual analysis in groups of patients.

Phenotypic profile of peripheral blood lymphocytes in patients with inflammatory bowel diseases

Mechanisms of recognition and effector responses of immune system upon initiation and maintenance of immune-mediated inflammation and tissue damage in inflammatory bowel diseases, including Crohn’s disease and ulcerative colitis (UC), have been actively studied over recent time. Existing evidence suggests these diseases to be caused by abnormal immune response against intestinal flora microorganisms in genetically susceptible individuals. Despite available data on the features of immune disorders in ulcerative colitis and Crohn’s disease, there are still many questions about the involved minor lymphocyte subpopulations and contribution of various functionally active molecules, which play a key role in recognition and initiation of the immune response and can be considered biomarkers of a pathological process in inflammatory bowel diseases. The populations of T lymphocytes with γδT cell receptor, B1 cells and NK T lymphocytes are of greatest interest, as well as functionally active TLRs (Toll-like receptors), CD89, CD314, etc. Due to substantial progress in studying the nature of recognition and activation of the immune cells, the paper presents phenotypic and functional characteristics of major and minor subpopulations of peripheral blood lymphocytes observed in 25 patients treated at the Surgery Department of the State Institution “Minsk Regional Clinical Hospital” (Republic of Belarus) from 2018 to 2020. The detected changes in peripheral blood lymphocyte phenotype of inflammatory bowel diseases patients suggest distinct immunological profiles prevailing in the damage mechanisms in Crohn’s disease and ulcerative colitis. I.e., in Crohn’s disease patients, B1 lymphocytes with CD19+CD5+ and NK cells in combination with increased CD56bright population, as well as NK T cells with anti-inflammatory and regulatory activity are involved into genesis of the disease. In ulcerative colitis, T, B, NK lymphocytes with pro-inflammatory phenotype and T lymphocytes with γδ T cell receptor may play a pathogenetic role in maintenance of chronic inflammation. With respect to functional significance of activating receptors, the number of TLR4- и CD89-positive cells may be used for developing immunological criteria/ biomarkers of therapeutic efficacy of new drugs. Studying interactions between innate and adaptive immunity will open new perspectives in understanding immunological disorders associated with chronic gastrointestinal inflammation.

Investigation of Erythema, Radiation Dose, and Radiation-Induced Apoptosis in the Peripheral Blood Lymphocytes of Patients Treated with Radiofrequency Catheter Ablation

Introduction: The long-term use of fluoroscopy in cardiac interventional procedures increases the patient dose and causes severe skin reactions, which lead to growing concern. The aim of the present study was to evaluate the risk and the effect of X-ray irradiation on apoptosis in the peripheral blood lymphocytes of patients treated with ablation in electrophysiological studies. Material and Methods: A total of 30 patients who underwent ablation therapy participated in this study. The absorbed dose in the given area was measured by a thermos luminescent dosimeter (TLD). The duration of dose delivery, absorbed dose by the apparatus, and dose area product (DAP) factor were measured for each patient. The skin changes were observed within the 1st day to 5th week after the operation. Blood sampling was conducted (before and 24 h after the treatment), and then, flow cytometry was performed to investigate the apoptotic changes in the blood lymphocytes. Results: The statistical analysis showed that there was a significant difference in the apoptosis of patient blood lymphocytes before irradiation and following that (p <0.05). There was a correlation between the amount of DAP and TLD dose (p <0.001). Furthermore, a correlation was observed between the total apoptosis and fluoroscopic time. The patient radiation dose in the ablation test was not in the threshold level required to create skin erythema. Conclusion: The results of the present study revealed that the use of long-time fluoroscopy in electrophysiological studies may cause a significant increase of apoptosis in the peripheral blood lymphocyte of patients treated using this procedure.

Correlation analysis of microRNA-126 expression in peripheral blood lymphocytes with apoptosis and prognosis in patients with sepsis

To analyze the relationship between the expression of microRNA-126 (miR-126) in peripheral blood lymphocytes with apoptosis and prognosis in patients with sepsis, and to explore its potential regulatory mechanism. Thirty patients with general infection and 20 patients with sepsis admitted to the department of intensive care unit (ICU) of the First Affiliated Hospital of Bengbu Medical College from January to December 2019 were enrolled. Peripheral blood was taken to separate lymphocytes, and the expressions of miR-126 and caspase-3 were detected by reverse transcription-polymerase chain reaction (RT-PCR). At the same time, the liver and kidney function and other laboratory indexes were measured, and the sequential organ failure assessment (SOFA) and acute physiology and chronic health evaluation II (APACHE II) scores were calculated. The 28-day prognosis was observed. Pearson method was used to analyze the correlation between miR-126 and caspase-3, APACHE II score. Receiver operating characteristic (ROC) curve was used to analyze the predictive value of miR-126 on prognosis; at the same time, according to the best cut-off value of miR-126 in predicting prognosis, the patients were divided into two groups, and the 28-day Kaplan-Meier survival curve was drawn. The expression of miR-126 in peripheral blood lymphocytes of patients with sepsis was lower than that of patients with general infection [miR-126 mRNA (2-ΔΔCt): 1.239±0.134 vs. 1.599±0.110, P < 0.01], while the expression of caspase-3 and APACHE II score were significantly increased [caspase-3 mRNA (2-ΔΔCt): 1.172±0.132 vs. 0.901±0.143, APACHE II: 19.75±3.74 vs. 12.63±3.94, both P < 0.01]. Pearson correlation analysis showed that the expression of miR-126 was negatively correlated with the expression of caspase-3 (r = -0.678, P < 0.001) and APACHE II score (r = -0.581, P < 0.001). ROC curve analysis showed that the area under the ROC curve (AUC) for predicting the prognosis by miR-126 expression in peripheral blood lymphocytes was 0.823 (P < 0.001). When the best cut-off value was 1.395, the sensitivity was 75.0%, the specificity was 71.4%, the positive predictive value was 81.1%, the negative predictive value was 63.6%, the positive likelihood ratio was 2.622, and the negative likelihood ratio 0.350. In addition, the patients were divided into high miR-126 group (miR-126 > 1.395, n = 31) and low miR-126 group (miR-126 ≤ 1.395, n = 19) according to the best cut-off value of miR-126. Kaplan-Meier survival curve analysis showed that the 28-day cumulative survival rate of high miR-126 group was higher than that of low miR-126 group (Log-Rank: χ2 = 11.702, P = 0.001). miR-126 in peripheral blood lymphocytes of patients with sepsis may affect immune status by promoting apoptosis of lymphocytes, and its expression level can reflect the severity and prognosis of sepsis.

Melatonin a Promising Candidate for DNA Double-Stranded Breaks Reduction in Patients Undergoing Abdomen-Pelvis Computed Tomography Examinations.

Cancer incidence is 24% higher in children and young adults exposed to Computed Tomography (CT) scans than those unexposed. Non-repairing of ionizing radiation-induced DNA Double-Strand Breaks (DSBs) can initiate carcinogenesis. In the present study, we aimed to investigate the radioprotective potential of melatonin against DSBs in peripheral blood lymphocytes of patients undergoing abdomen-pelvis CT examinations. This double-blind, placebo-controlled clinical trial was conducted on thirty patients. These patients were divided into two groups; group one (control) patients who have undergone the CT examination received a single oral dose of placebo, while in group two, patients received a single oral dose of 100mg melatonin. In both the groups, blood samples were collected 5-10min before and 30 minutes after the CT examination. The lymphocytes from these samples were isolated and DSBs were analyzed using γH2AX immunofluorescence microscopy. Compared to the control group, the use of melatonin 1h before the CT examination caused a significant reduction in γH2AX-foci, indicating a reduction in DSBs. In addition, no side effect was observed in patients following 100mg melatonin administration. For the first time, this study has shown that melatonin has protective effects against radiationinduced genotoxicity in peripheral blood lymphocytes of patients undergoing abdomen-pelvis CT examinations. Therefore, melatonin can be considered as a promising candidate for reducing DSBs in patients undergoing abdomen-pelvis CT examinations.

Overexpression of PD-1 on Peripheral Blood Lymphocytes in Patients with Idiopathic Pulmonary Arterial Hypertension and Its Association with High Viral Loads of Epstein-Barr Virus and Poor Clinical Parameters.

Idiopathic pulmonary arterial hypertension (IPAH) is a rare but severe disease with the elevated blood pressure in the pulmonary arteries without a known trigger of vascular remodelling. It leads to the right heart failure with reduced survival. Changes in the immunological landscape of the lungs and the periphery are common in IPAH patients, suggesting an immune system dysfunction. A cohort of 25 IPAH patients was enrolled in our study to investigate a link between the patient’s clinical status, immune parameters of the blood, and the Epstein–Barr virus (EBV) infection. We found significant alterations of the patients’ peripheral blood parameters. Therein, T lymphocytes and NK cell counts were decreased in the IPAH patients’ blood, while the proportion of regulatory T cells was increased. Additionally, levels of proinflammatory cytokines interleukin-6 (IL-6), IL-2, and interferon-gamma (IFN-γ) were elevated. We identified a weak correlation between EBV loads and IPAH patients’ clinical state (r = 0.54) and between EBV loads and overexpression of PD-1 on helper T cells (r = 0.56). We speculate that a significant dysregulation of the immune system homeostasis observed in IPAH patients may contribute to increased susceptibility of those patients to EBV infection, yet further longitudinal studies are required to characterize this relation in detail.

P0454MORPHODENSITOMETRIC FEATURES OF PERIPHERAL BLOOD LYMPHOCYTES IN PATIENTS WITH PRIMARY GLOMERULONEPHRITIS

Abstract Background and Aims Lymphocytes play an important role in the development of glomerulonephritis (GN). Their subpopulations are activated under the influence of certain cytokines that act on the activation factors of transcription of these cells, leading to the corresponding regulation of gene expression. However, in addition, in the regulatory processes of lymphocyte functions, epigenetic modifications of the degree of nuclear chromatin compaction are important. The morphological manifestation of the latter may be the state of the optical density of the nuclei and the range of its variability due to the ratio of chromatin condensation/decondensation. The study of the features of potentially reversible changes in chromatin structure in somatic cells becomes actual. One of the convenient objects for studying the epigenetic mechanisms of the genome functioning regulation are interphase nuclei of peripheral blood lymphocytes. Morphodensitometric study permits to establish compensatory changes at the cellular level and to evaluate the functional state of the hereditary apparatus. The aim of the research was to study the morphodensitometric features of genome status of peripheral blood lymphocytes in patients with primary GN. Method We examined 119 patients (76 men and 43 women) with primary GN. The average age of patients was (39.65±14.16) years. The patients underwent a renal biopsy to verify the morphological form of the disease. For comparison, a survey of 30 healthy individuals was conducted. Chromatin of nuclei and its changes were studied using an image analyzer based on the programm Image Tool for Windows (v. 3.0). The advantage of this method is the possibility of a combined assessment of morphometric (area, perimeter), spectrometric (optical and integral density) data with the analysis of the microanatomy of isolated chromatin blocks and the chromatin function at the same time. The study of the morphodensitometric characteristics of peripheral blood lymphocytes is an informative and accessible method for assessing the general state of the function and structure of the hereditary apparatus of the organism. Results The sexual characteristics of the morphometric and optical parameters of lymphocytes nuclei of patients were established. So, in all patients and especially women, a decrease in the perimeter of lymphocytes nuclei was registered in 1.25 and 1.31 times, respectively (p &amp;lt;0.05). The area of these cells nuclei also naturally decreased in 1.22 times in all patients (in 1.08 and 1.34 times in men and women, respectively) compared to the control (p&amp;lt;0.05). A significant decrease in the area of lymphocytes nuclei with an unchanged density of chromatin packing in these nuclei limits the space inside the nucleus for the interaction of individual chromosome loci in this space. Such limitations can be a serious obstacle to the normal functioning of the genome and its repairing processes. Meanwhile, differences in the optical density parameter of lymphocytes nuclei in both groups were defined. The minimum and maximum optical density were higher in the group of patients, and the range of optical density variability showed wider borders of functional heterogeneity of cells compared to the control. Conclusion Comparative analysis of morphodensitometric features of peripheral blood lymphocytes in patients with primary GN detected the changes in the ratio of chromatin condensation/decondensation and normal structure of nuclei, that may indicate functional disorders of the genotype due to exo- and endogenous factors.

Genomic instability in peripheral blood lymphocytes of patients diagnosed with high-grade squamous intraepithelial lesions: CIN 2 versus CIN 3

Cancer is a genomic disease associated with accumulation of genetic damage. Cancer-initiating events, such as chromosome breakage, loss and rearrangement, can be used as biomarkers to evaluate individual cancer risk. Cytokinesis-block micronucleus cytome (CBMN - Cyt) assay parameters in peripheral blood lymphocytes (PBL) of thirty four patients diagnosed with high-grade squamous intraepithelial lesions (HSIL) and fifteen healthy women were measured. The genomic instability of patients diagnosed with HSIL were investigated in order to compare differences between the two subgroups of HSIL (CIN 2 and CIN 3). The micronucleus (MN) frequencies in PBL, as well as the frequencies of nucleoplasmic bridges (NPB) and nuclear buds (NBUD) were higher in patients than in controls (Mann- Whitney test, p < 0.05). These results provide evidence that CBMN cytome assay in peripheral blood lymphocytes may be used to identify individuals who are at high risk of developing cervical cancer. Since the extent of DNA damage varies between CIN 2 and CIN 3, these findings support the CIN grading system.

Expression of CD25+ and CD95+ Surface Markers on Peripheral Blood Lymphocytes in Patients with Vibration Disease

Vibration disease occupies one of the leading places among occupational diseases. The development and course of vibration disease is accompanied by disorders in immune system. Aim of the study is to provide a comparative assessment of cells with markers of the early (CD25+) and late (CD95+) stage of lymphocyte activation and to compare them with the phenotypic composition of lymphocytes in patients with vibration disease. Materials and methods . The study includes men aged 36-60 years with vibration disease from exposure to local vibration (26 people) and from combined exposure to local and general vibration (28 people). Results . The most pronounced changes in the system of lymphocyte apoptosis are observed in patients with vibration disease from the combined effects of local and general vibration, manifested by the increase in the number of CD25+ and CD95+-lymphocytes. Increase of quantity of CD25+ at patients with a vibration disease from impact of local vibration and from the combined impact of local and general vibration is caused by increase in their number on lymphocytes what the direct correlation dependence between quantity of CD25+-lymphocytes and the number of CD3+, CD4+, CD8+, CD16+, CD20+-lymphocytes testifies to. Conclusions . In patients with vibration disease from the combined effects of local and general vibration, the increase in the number of CD95+ is due to an increase in their density on mature T-lymphocytes, as evidenced by the inverse relationship between the level of CD95+ and the CD3+-lymphocytes. The obtained data should be taken into account in order to develop new effective methods of prevention, treatment and prediction of the course of vibration pathology.