Peripheral Blood Lymphocytes Research Articles

The decreased of peripheral blood natural killer cell is associated with serum IL-2 level in the renal tubular acidosis in patients with primary sjogren’s syndrome

BackgroundPrimary Sjogren’s Syndrome (pSS) is a lymphoproliferative disease with autoimmune characteristics, which is characterized by lymphocyte infiltration of exocrine glands and involvement and dysfunction of extraglandular organs. Renal tubular acidosis (RTA) is a common renal involvement in pSS. This study investigated the phenotypic characteristics of peripheral blood lymphocyte subsets and cytokines in pSS patients complicated with RTA (pSS-RTA).MethodThis retrospective study included 25 pSS patients complicated with RTA and 54 pSS patients without RTA (pSS-no-RTA). To examine the level of peripheral lymphocytes subsets, flow cytometry analysis was used. The level of serum cytokines were detected by flow cytometry bead array(CBA). The influencing factors related to the occurrence of pSS-RTA were identified through logistic regression analyze.ResultsThe absolute number of CD4 + T cells and Th2 cells in peripheral blood were decreased in pSS-RTA patients than pSS-no-RTA patients. Moreover, the absolute number of NK cells and Treg cells were also decreased in pSS-RTA patients than pSS-no-RTA. The level of serum IL-2 was higher in pSS-RTA patients than pSS-no-RTA patients, and is negatively correlated with the number of NK cells, the number and percentage of Th17 cells, and Th17/Treg. Serum IL-2 level is also correlated with various cytokines. Multivariate logistic analysis proved that elevated ESR and ALP were risk factors for pSS complicated with RTA, while Treg was a protective factor.ConclusionThe increase of serum IL-2 level and the decrease of peripheral blood NK cells and Treg cells may be the immune mechanism of the development of pSS-RTA disease.

QUANTIFICATION OF DNA DOUBLE-STRAND BREAKS IN BENIGN AND MALIGNANT BREAST DISEASES

Relevance: Double-strand DNA breaks are the most dangerous DNA damage. Analysis of foci of phosphorylated histone protein H2AX (γH2AX) is currently the most sensitive method for detecting DNA double-strand breaks. This protein modification can become a biomarker of cellular stress, especially in diagnosing and monitoring neoplastic diseases. In this study, we utilized novel pattern recognition algorithms on the AKLIDES® platform to automatically analyze immunofluorescent images of γH2AX foci and compare the results with visual scores. The γH2AX foci formation on peripheral blood mononuclear cells of women with breast cancer or benign breast tumors was studied.
 The article aimed to quantify DNA double-strand breaks in peripheral blood lymphocytes in women with breast cancer and benign breast masses to identify a possible biomarker.
 Methods: γ-H2AX foci in lymphocytes were analyzed using the automated AKLIDES system in patients with breast cancer (n=29) and benign breast tumors (n=24).
 Results: When comparing the indicators of the main and control groups in the channel of ruptures “FITC,” a statistically significant difference was found in the indicators “Foci dia” (p=0.0382), “Focilnt mean” (p=0.0166), “Colocalisation” (p=0.0486). In the repair channel “AРС,” significant differences were found in the indicators “Nuclei BGInt” (p=0.0166) and the indicator “Focilnt mean” (p=0.0118).
 Conclusion: The revealed changes of DNA double-strand breaks along the FITC break channels and APC repair between the main and control groups can possibly serve as a breast cancer diagnostic marker.

DNA DOUBLE-STRANDED BREAKS AND REPAIRS IN ACUTE LEUKEMIA

Relevance: Double-strand Relevance: Errors in the damage repair system, such as double-stranded DNA breaks, can lead to mutations that will be passed on to subsequent generations of cells, and some of these mutations may have oncogenic potential.
 The study aimed to evaluate the number of double-stranded breaks and DNA repairs of peripheral blood lymphocytes in a group of conditionally healthy children and in patients diagnosed with acute leukemia (AL) to develop a method for predicting the outcome of the disease and determining the effectiveness of therapy.
 Methods: peripheral blood lymphocytes were studied: a) 38 conditionally healthy children (control group); b) 100 patients diagnosed with acute leukemia (AL); c) 14 children with relapse of the disease. Double-stranded DNA breaks/repairs were examined using the Aklides system (MEDIPAN, Germany), consisting of a fluorescent analyzer and the AKLIDES Nuk software.
 Results: In patients with T-lymphoblastic leukemia, both at admission and the end of Day 7 at the hospital, the number of 53BPI repair foci was, on average, three times higher than the number of DNA damages. In most cases, the ratio of breaks/repairs indicators during treatment did not change among patients with B-line leukemia. Double-stranded DNA breaks prevailed over repairs, with the newly established disease on the 7th, 15th day, and 3rd month of treatment.
 Overall, 22 samples were tested using RT-PCR, and results were considered as follows:1 (4%) – positive, 13 (59%) – negative, 8 (37%) -– invalid.
 Conclusion: The level of lymphocyte DNA damage in patients with B-ALL was higher than expected. In addition, the ratio of double-strand breaks to repairs remained unchanged at all stages of therapy in patients with B-ALL. The changes we suggest in these patients can be observed during and/or after maintenance therapy. Monitoring double-strand breaks/reparations was the initial step in developing a method of predicting the disease outcome and determining the therapy efficacy. The results obtained are of direct interest and require further research.

Subcutaneous Injection of Allogeneic Adipose-Derived Mesenchymal Stromal Cells in Psoriasis Plaques: Clinical Trial Phase I.

Mesenchymal stromal cells (MSCs) play immunomodulatory role in various autoimmune diseases. Previous pre-clinical and clinical studies have shown that MSCs could be a therapeutic modality for psoriasis. However, the mechanisms of treatment and its possible side effects are under investigation. In this study, the safety and probable efficacy of injecting allogeneic adipose-derived mesenchymal stromal cells (ADSCs) in psoriatic patients were evaluated. In this phase I clinical study with six months of follow-up, total number of 1×106 or 3×106 cells/cm2 of ADSCs were injected into the subcutaneous tissue of each plaque as a single dose in three males and two females (3M/2F) with a mean age of 32.8 ± 8.18. The primary outcome was safety. Changes in clinical and histological indexes, the number of B and T lymphocytes in local and peripheral blood, and serum levels of inflammatory cytokines were assessed. Paired t test was used to compare variables at two time points (baseline and six months after injection) and repeated measures ANOVA test was utilized for variables at three time points in follow-up visits. No major adverse effects such as burning, pain, itching, or any systemic side effects were observed following ADSCs injection, and the lesions showed slight to considerable improvement after injection. The mRNA expression levels of pro-inflammatory factors were reduced in the dermis of the patients after injection. The increased expression level of Foxp3 transcription factor in the patient blood samples suggested modulation of inflammation after ADMSCs administration. Six months after the intervention, no major side effects were reported, but skin thickness, erythema, and scaling of the plaques, as well as the PASI score, were decreased in majority of patients. Our study suggested that ADSC injection could be considered as a safe and effective therapeutic approach for psoriatic plaques (registration number: IRCT20080728001031N24).

Systemic production of cytokines and growth factors in rats with metabolic syndrome

ABSTRACT. An important characteristic of the state of the immune system is the production of cytokines, which determine the development of metabolic processes and are aimed at the immune response. Changes in the immune response occur with the development of homeostasis system disorders that change the rheological characteristics of the blood and contribute to the development of the metabolic syndrome (MS). The aim of the study was to study changes in the functional activity of cytokines and angiogenic growth factors (GF) during experimental MS in rats depending on age. Мaterials. MS modeling was carried out on 21 rats, which were divided into 3 groups, 7 animals each, of the same age (1a subgroup - 6 months of age, 2a subgroup - 9 months of age, 3a subgroup - 12 months). In the pathogenesis of the development of MS The role of MS in the development of systemic production of cytokines and FR has been studied, and data on the content of pro-inflammatory cytokines in the peripheral blood depending on the age of rats have been presented. The results of the study of the content of growth factor (GF) and endothelin in peripheral blood in MS tended to decrease with increasing age of rats providing morpho- and angiogenesis (p<0.01). An increase in the concentration of endothelin in the blood serum of rats with MS may contribute to the activation of vascular endothelial cells and an increase in the level of fibrinoid in the vascular system with increasing age. The level of cytokines circulating in the peripheral blood in rats with MS significantly differed from that, which was manifested by an increase in the level of IL-4, IL-8 and IL-10. Spontaneous production of cytokines by peripheral blood lymphocytes in MS increased with the age of rats, which indicated an increase in the secretion of IL-6 and IL-10, and a decrease in the production of IFN-1 and TNF-α. The decrease in spontaneous production of IL-1β and IL-10 in 24-hour cultures of peripheral blood monocytes in rats was significantly higher compared to IL-6 (p<0.001) compared to the 2nd and 3rd - observation groups. The content of IL-8 and TNF-α did not differ significantly (p>0.05), and IL-12 was below the sensitivity limit of the test system (less than 3 pg/ml). Conclusion. The data obtained indicate that in the peripheral blood of rats with MS, the secretion of pro-inflammatory and regulatory cytokines, as well as FR, decreases with age. Hormones also have an inhibitory effect on the production of cytokines by macrophages, the level of which changes significantly during MS. The results of the study indicate various pathogenetic mechanisms of the formation of MS in rats, accompanied by a violation of the immune response of mononuclear cells and angiogenic RF at the system level.

Association of MTHFR gene polymorphism in preeclampsia and recurrent pregnancy loss: A case-control study from South India

BackgroundPreeclampsia (PE) and recurrent pregnancy loss (RPL), both of which affect maternal and reproductive health, are obstetrical-gynecological issues. Pregnancies that meet one or more of these criteria are considered high risk. This study aims to examine and understand how specific methylation within the MTHFR gene (rs 1,801,131 and rs 1,801,133) polymorphisms may contribute to preeclampsia and recurrent miscarriages in the South Indian population. MethodsA combination of recurrent pregnancy loss and preeclampsia were taken as a case (146), and healthy controls (152) were enrolled in the study. 5 ml of human blood was taken after getting consent from the participants. The DNA was isolated from the peripheral blood lymphocytes and genotyped using ARMS-PCR. ResultThe findings of this study suggest that the MTHFR gene (rs1801131) is not associated with PE and RPL risk. A significant association has been observed between rs1801133 and the derived allele T (OR = 0.6, 95% CI -0.4-0.8, p = 0.001). Furthermore, a p-value of 0.01 was calculated for the homozygous recessive genotype TT (OR = 0.5, 95% CI - 0.29-0.1). ConclusionsThe results of this study suggest that preeclampsia and recurrent pregnancies are more common among South Indian women who carry a variant of the MTHFR gene 1,801,133. According to the conclusion, higher sample sizes and a wider study are necessary to verify these findings. More diverse and valuable information can be obtained through large sample sizes and combined studies, allowing the results to be applied to a wide population.

The impact of adjuvanted and non-adjuvanted influenza vaccines on <i>in vitro</i> lymphocyte immunophenotype

Vaccination is the most effective method of influenza prophylaxis resulting in reduced frequency and severity of complications. Currently, for the prevention of influenza, inactivated split and subunit vaccines as the safest and promoting formation of protective level of strain-specific virus neutralizing antibodies are used. It is known that not all inactivated vaccines are effective enough for select human groups. While nowadays the level of public health is low, there is a need to improve the effectiveness of vaccines that should activate all chains of the immune system. In order to enhance intensity of influenza virus strain-specific antibody production, adjuvant vaccines exerting other mechanisms to activate humoral and cellular immunity compared to non-adjuvant vaccines have been used. The aim of the study was to examine lymphocyte immunophenotype in 27 healthy donors treated with polymer-subunit (immunoadjuvant) and non-adjuvanted split and subunit influenza vaccines.
 Materials and methods. Peripheral blood lymphocyte subpopulations were studied in vitro by flow cytometer FC-500 Cytomics (Beckman Coulter, USA) using FITC- and PE-labeled monoclonal antibodies (mAbs).
 Results. All examined influenza vaccines activate the effectors of cellular immunity, increasing the number of NK-cells (CD16/56), NKT-lymphocytes (CD3/CD16/56), B-lymphocytes (CD45/CD20), activated (CD3/HLA-DR) and cytotoxic (CD8/HLA-DR) T-lymphocytes, as well as cells bearing early activation marker (CD45/CD25). Among them the immunoadjuvant vaccine showed the greatest potential to induce cellular response eliciting regulatory mechanisms that prevent hyperactivation, stimulating growth of NK (CD16/56), NKT-cells (CD3/CD16/56), B-lymphocytes (CD45/CD20), activated (CD3/HLA-DR) and cytotoxic (CD8/HLA-DR) T-lymphocytes, T-regulatory cells (Tregs, CD4/CD25/Foxp3).
 Conclusion. Vaccination against influenza besides the formation of specific antibodies render a transient, immunomodulating effect that is more noticeable after immunoadjuvant vaccine. It can be assumed that vaccination of people with dysfunctions of the immune system an additional prophylactic effect will be observed.

P-352 Evaluation of CD25 and CD69 activation markers expression on peripheral blood cells subpopulations in endometriosis patients

Abstract Study question We aim to determine the percentages of CD25-positive and CD69-positive lymphocytes in peripheral blood of endometriosis (EMS) patients. Summary answer Higher percentages of lymphocytes expressing CD69 and CD25 markers in EMS and their correlation with the severity of the disease, indicate persistent activation of lymphocytes What is known already Exposure to antigens causes activation markers to appear on the surface of lymphocytes. Among them, we can distinguish early and late activation markers. The earliest one is CD69, which is expressed upon activation by the TCR receptor. CD69 plays a role in the proliferation and survival of activated T lymphocytes. CD25 is a moderate late activation marker, considered the most important marker of cellular response activation. The significance of CD69 and CD25 expression by T cells in endometriosis has not yet been determined. Increased CD69 expression on various lymphocyte subsets in peritoneal fluid in EMS has been reported. Study design, size, duration Within this prospective study between January 2016 and August 2018 immune diagnostics of the number of CD25-positive and CD69-positive lymphocytes in peripheral blood of EMS patients (firstly diagnosed during laparoscopy and confirmed histopatologically) and non-EMS controls were performed using flow cytometry analysis. Total number of 74 subjects were included in the study. All patients signed written informed consent before their enrollment in the study. Participants/materials, setting, methods We enrolled 54 subjects with previously untreated endometriosis and 20 healthy age-matched controls. Peripheral blood was collected from control patients to assess the immunophenotype of lymphocytes and measure expression of activation markers Cd25 and CD69 on several subtypes B and T lymphocytes. Diagnosis and assessment of the stage of the EMS was established during laparoscopy using rASRM score. Differences were considered statistically significant with a p < 0.05. Main results and the role of chance Significantly higher expression of the CD25 and CD69 antigen was found in both CD3+ T, CD4+ T and CD8+ T cells as well as CD19+ B cells. In each of the studied groups, a significance level of p < 0.001 was obtained. There was a weak positive correlation between the percentage of CD4+CD25+ T cells and the stage of endometriosis (R = 0.357; p = 0.008) and negative correlations between the percentage of endometriosis and the percentage of CD3+CD69+ T cells (R = −0.554; p < 0.001), T CD4+CD69 + (R = −0.554, p < 0.001) and T CD8+CD69 + (R = −0.553, p < 0.001). The expression of activation markers, CD25 and CD69 antigens in patients suffering from endometriosis with accompanying clinical symptoms was also evaluated. n the group of patients with endometriosis accompanied by infertility or pelvic pain, no statistical differences in the expression of CD25 and CD69 antigens were observed. The only clinical condition coexisting with statistically different expression of activation markers is adhesion disease accompanying endometriosis - a statistically significantly lower percentages of T CD3+CD69 + (p = 0.002), T CD4+CD69 + (p = 0.002) and T CD8+CD69 + (p = 0.002). Limitations, reasons for caution Small sample size is an important limitation of this study. In addition, evaluating immunological analysis only in serum samples does not let us drive any conclusions on the local changes of endometriosis lesions. Wider implications of the findings Clinical interpretation of changed expression of CD69 and CD25 in EMS could be useful in evaluating mechanisms of cellular activation, peripheral tolerance and immune imbalance involved in pathogenesis of endometriosis. Further studies to understand mechanisms underlying correlation between stage of the disease and expression of CD69 should be considered. Trial registration number not applicable

Self-assembled nanoparticle with E protein domain III of DTMUV based on ferritin as carrier can induce a more comprehensive immune response and against DTMUV challenge in duck

Duck Tembusu virus (DTMUV) causes severe reduction in egg production and neurological symptoms in ducklings. Vaccination is the primary measure used to prevent DTMUV infections. In this study, self-assembled nanoparticles with the E protein domain III of DTMUV, using ferritin as a carrier (EDⅢ-RFNp), were prepared using a prokaryotic expression system. Ducks were intramuscularly vaccinated with EDⅢ-RFNp, EDⅢ protein, an inactivated vaccine HB strain (InV-HB), and PBS. At 0, 4, and 6 weeks post-primary vaccination, the EDIII protein-specific antibody titre, IL-4, and IFN-γ concentrations in serum were determined by ELISA, and neutralising antibodies titres in sera were determined by virus neutralising assay. Peripheral blood lymphocytes proliferation was determined by CCK-8 kit. Following challenge with the virulent DTMUV strain, the clinical signals and survival rate of the vaccinated ducks were recorded, and DTMUV RNA levels in the blood and tissues of the surviving ducks were determined by real-time quantitative RT-PCR. The near-spherical EDⅢ-RFNp nanoparticles with 13.29 ± 1.43 nm diameter were observed by transmission electron microscope. At 4 and 6 weeks post-primary vaccination, special and Virus neutralisation (VN) antibodies, lymphocyte proliferation (stimulator index, SI), and concentrations of IL-4 and IFN-γ in the EDⅢ-RFNp group were significantly higher than in the EDⅢ and PBS groups. In the DTMUV virulent strain challenge test, the EDⅢ-RFNp-vaccinated ducks showed milder clinical signs and higher survival rates than EDⅢ- and PBS-vaccinated ducks. The DTMUV RNA levels in the blood and tissues of EDⅢ-RFNp-vaccinated ducks were significantly lower than those in EDⅢ- and PBS-vaccinated ducks. Additionally, the EDⅢ protein-special and VN antibodies, SI value, and concentration of IL-4 and IFN-γ in the InV-HB group was significantly higher than that of the PBS group at 4 and 6 weeks post-primary vaccination. InV-HB provided more efficient protection than PBS based on a higher survival rate, milder signals, and lower levels of the DTMUV virus in the blood and tissues. These results indicated that EDⅢ-RFNp effectively protected ducks against DTMUV challenge and could be a vaccine candidate to prevent DTMUV infection.

Comparison of comet-based approaches to assess base excision repair

DNA repair plays an essential role in maintaining genomic stability, and can be assessed by various comet assay-based approaches, including the cellular repair assay and the in vitro repair assay. In the cellular repair assay, cells are challenged with a DNA-damaging compound and DNA damage removal over time is assessed. In the in vitro repair assay, an early step in the repair process is assessed as the ability of a cellular extract to recognize and incise damaged DNA in substrate nucleoids from cells treated with a DNA-damaging compound. Our direct comparison of both assays in eight cell lines and human peripheral blood lymphocytes indicated no significant relationship between these DNA repair assays (R2 = 0.084, P = 0.52). The DNA incision activity of test cells measured with the in vitro repair assay correlated with the background level of DNA damage in the untreated test cells (R2 = 0.621, P = 0.012). When extracts were prepared from cells exposed to DNA-damaging agents (10 mM KBrO3 or 1 µM Ro 19–8022 plus light), the incision activity was significantly increased, which is in line with the notion that base excision repair is inducible. The data presented suggest that the two assays do not measure the same endpoint of DNA repair and should be considered as complementary.

Effect of Nasal Irrigation in Children With Omicron Variant of COVID-19 Infection.

Objective: To explore the effect of nasal irrigation on the disappearance of symptoms and nucleic acid conversion in children with Omicron variant. Methods: This quasi-experimental study included children diagnosed with asymptomatic, mild, and moderate Omicron variant infection during the isolation observation period in the Shandong Public Health Clinical Center between April 1, 2022 and May 1, 2022. The children were divided into a routine group (received Lianhua Qingwen (LhQw) Granules), isotonic saline group (received LhQw Granules combined with isotonic saline nasal irrigation), and hypertonic saline group (received LhQw Granules combined with 3% hypertonic saline nasal irrigation), respectively. The primary outcomes were the time of symptom disappearance and nucleic acid conversion time. The secondary outcomes were peripheral white blood cell count (WBC), lymphocyte count (LYM), neutrophil count (NEU), and C-reactive protein (CRP) levels. Results: A total of 60 children (7.26 ± 3.15years old) were included (20 per group). The average time of nucleic acid conversion in the 2 saline nasal irrigation groups was significantly reduced compared with the routine group (all P < 0.001), while the fever time and cough duration among the 3 groups were comparable (all P > 0.05). LYM count in the 2 saline nasal irrigation groups was significantly increased after treatment compared to before treatment and was significantly higher than in the routine group (all P < 0.05). There was no significant difference in LYM count between the isotonic and hypertonic saline groups (P = 0.76). Additionally, all children in the saline group well tolerated the treatment, and no adverse events occurred in the isotonic saline group. Conclusions: Timely use of saline nasal irrigation may promote nucleic acid conversion in children with Omicron virus infection.

Oxidative and DNA damage in obese patients undergoing bariatric surgery: A one-year follow-up study

The pathogenesis of obesity and related comorbidities has long been associated with oxidative stress. The excess of adipose tissue contributes to the production of free radicals that sustain both a local and a systemic chronic inflammatory state, whereas its reduction can bring to an improvement in inflammation and oxidative stress. In our work, using the fluorescent lipid probe BODIPY® 581/591 C11 and the γH2AX foci assay, a well-known marker of DNA double strand breaks (DSB), we evaluated the extent of cell membrane oxidation and DNA damage in peripheral blood lymphocytes of normal weight (NW) controls and obese patients sampled before and after bariatric surgery. Compared to NW controls, we observed a marked increase in both the frequencies of oxidized cells or nuclei exhibiting phosphorylation of histone H2AX in preoperatory obese patients. After bariatric surgery, obese patients, resampled over one-year follow-up, improved oxidative damage and reduced the presence of DSB. In conclusion, the present study highlights the importance for obese patients undergoing bariatric surgery to also monitor these molecular markers during their postoperative follow-up.

Effect of radiation emitted from mobile phone on bone marrow haematopoietic stem cell in mice

The present research aims to explore the potential impact of mobile phone radiation on bone marrow haematopoietic stem cell in mice. 160 male BALB/C mice aged 3–4 weeks were randomly distributed into 4 groups as blank control, control, TD-SCDMA and LTE-Advanced respectively, with 40 mice in each group. Mice were designed to receive cell phone radiation for 2–8 weeks. 10 mice in each group were taken out for examinations given radiation exposure periods were 2, 4, 6 and 8 weeks respectively. Then terminal deoxynuclotidyl transferase-mediated dUTP Nick-end labelling technique was conducted to detect the apoptosis of stem cell, and morphological method was carried out for the examination of micronucleus and subpopulations of bone marrow haematopoietic stem cell, while enzyme immune technique was performed to analyse the subsets of T lymphocyte. As a result, no statistical differences were discovered on issues of apoptosis and micronucleus during the entire experimental period in the 2 sham radiation control groups. Notwithstanding similar results were seen in TD-SCDMA and LTE-Advanced groups after exposed to radiation for 2 or 4 weeks, apoptosis began to be enhanced and micronucleus to be increased given the mice experience longer exposure as 6 or 8 weeks. There were virtually negligible effects on subpopulations of stem cell, as well as on subsets of T lymphocyte of peripheral blood during the entire experimental period in the 4 groups. It could be concluded that long-term radiation from cell phone could induce apoptosis of haematopoietic stem cell in mice; moreover, it seems to stimulate the formation of micronucleus, yet with even negligible effect on the proportions of stem cell subpopulations and peripheral blood T lymphocyte subsets.

Bioinformatics analysis of lncRNA-related ceRNA networks in the peripheral blood lymphocytes of Kazakh patients with essential hypertension in Xinjiang.

Here, we aimed to investigate long non-coding RNA (lncRNA) expression characteristics in the peripheral blood lymphocytes of Xinjiang Kazakh people with essential hypertension and the underlying regulatory mechanisms of competing endogenous RNAs (ceRNA). From April 2016 to May 2019, six Kazakh patients with essential hypertension and six Kazakh healthy participants were randomly selected from the inpatient and outpatient cardiology departments of the First Affiliated Hospital of Shihezi University Medical College, Xinjiang. After detecting the expression levels of lncRNA and mRNA in the peripheral blood lymphocytes using gene chip technology, their levels in the hypertensive group were compared with those in the control group. Six differentially expressed lncRNAs were randomly selected for real-time PCR to verify the accuracy and reliability of the gene chip results. GO functional clustering and KEGG pathway analyses were performed for differentially expressed genes. The ceRNA regulatory network of lncRNA-miRNA-mRNA was constructed, followed by visualization of the results. The expressions of miR-139-5p and DCBLD2 after PVT1 overexpression in 293T cells were detected by qRT-PCR and Western blot. In the test group, 396 and 511 differentially expressed lncRNAs and mRNAs, respectively, were screened out. The trend of real-time PCR results was consistent with that of the microarray results. The differentially expressed mRNAs were found to be primarily involved in the adhesion spot, leukocyte migration via endothelial cells, gap junction, actin cytoskeleton regulation, and extracellular matrix-receptor interaction signaling pathways. By constructing the ceRNA regulatory network, we found that lncRNA PVT1-miR-139-5p-DCBLD2 has a potential ceRNA regulatory mechanism involved in the development of essential hypertension in Xinjiang Kazakh people. In 293T cells, lncRNA PVT1 overexpression inhibited miR-139-5p and DCBLD2 levels. Our findings indicate that differentially expressed lncRNAs may be involved in the development of essential hypertension. lncRNA PVT1-miR-139-5p-DCBLD2 was indicated to comprise a potential ceRNA regulatory mechanism involved in the development of essential hypertension in the Xinjiang Kazakh population. Thus, it may act as a novel screening marker or therapeutic target for essential hypertension in this population.

#2938 DE NOVO DSA IS ASSOCIATED WITH HUMAN PERIPHERAL BLOOD CXCR5+PD-1+CD8+ T CELLS AND PD-1 PD-L1 MRNA EXPRESSION IN KIDNEY TRANSPLANT PATIENTS

Abstract Background and Aims Donor-specific antibody (DSA) development is mainly responsible for chronic antibody-mediated rejection. The immunological events that cause the development of DSA is not fully elucidated. The relationship between the Follicular Cytotoxic T cell subgroup and the development of DSA is unknown. In this study we aimed to examine CD8+CXCR5+PD-1+ follicular cytotoxic T cell levels and expression levels of PD1/PD-L1 genes in peripheral blood lymphocytes in recipients who de-novo DSA positive or negative after kidney transplantation (KTX). Method In this study, the expression levels of PD-1, PD-L1 genes were determined using the Real-Time Quantitative PCR method from peripheral blood samples of 22 KTX patients with de-novo DSA positive, 20 KTX patients with DSA negative and 21 healthy control (Table 1). Our patient groups were PRA negative before transplantation. Expression (%) levels of target cells were evaluated by flow cytometry method. IBM SPSS Statistics for Windows Version 22 and R.3.3.2 software were used to evaluate the data. Results PD-1 expression level was higher in the KTX patients with de-novo DSA positive compared to the KTX patients with DSA negative group (p:0.006 CT: 0,84±0,23). According to the results of flow cytometry, CD8+CXCR5+PD-1+ cell expression (%) levels were found to be significantly higher in patients (3,06±1,98) compared to the transplant control group (0,52±0,40) (p: 0.001) (Figure 1). Conclusion It shows that there may be a direct correlation between DSA and PD-1 / PDL-1 mRNA level and CXCR5+PD1+CD8+ follicular cytotoxic T cell in transplant patients after kidney transplantation.

#5903 CLINICAL OUTCOME CORRELATION AMONG NEWLY DIAGNOSED LUPUS NEPHRITIS IN RELATION TO PERIPHERAL BLOOD LYMPHOCYTE SUBSET: A PROSPECTIVE STUDY

Abstract Background and Aims Given the heterogeneity in presentation, treatment practices and the outcome of lupus nephritis, there is a need for new protocols to predict outcome and rationalize treatment. Aim To prospectively study the correlation between peripheral blood lymphocyte subset, inflammatory markers and clinical outcomes among newly diagnosed lupus nephritis patients. Method Lymphocyte subset typing by flow cytometry method done at diagnosis and end of 6 months. SLEDAI -2k score. Intensive phase of Immunosuppression individualized by treating physician. Urine protein excretion was used to assess the final treatment outcome. Statistical analyses done using SPSS software. Two-sided P value of &amp;lt; 0.05 was considered significant. Pearson correlation coefficient formula used for correlations. Logistic regression done to identify factors responsible for clinical remission achieved at six months. Inclusion: All newly diagnosed renal biopsy proven Class 3, class 4, Class 5 or a combination class of lupus nephritis patients seen at Kasturba Hospital and Medical College, Manipal. Exclusion: Patients with active/recent infection, active malignancy, those with prior explosion to immunosuppression. Outcome measures, 1. To detect correlation if any, between peripheral blood lymphocyte subset, serum inflammatory markers and disease activity score at presentation. 2. To determine correlation if any, between peripheral blood lymphocyte subset and clinical remission achieved following immunosuppressive regimens at the end of six months of initial intensive treatment phase. Results 22 patients were taken for study. Relationship between SLEDAI score, lymphocyte subset characters, and clinical outcome given in Table 1 and 2. There was good correlation between lymphocyte subset characters, inflammatory markers and SLEDAI score, indicating ability of lymphocyte subset characters to characterize the severity of lupus nephritis. We found significant difference in lymphocyte subset characters between baseline and at the end of 6 months treatment. But there was no significant correlation for remission attainment with lymphocyte subset characters and SLEDAI score. Conclusion 1. Correlation found between peripheral blood lymphocyte subset, serum inflammatory markers and disease activity score at presentation and 6 months of treatment of lupus nephritis. 2. Peripheral blood Lymphocyte subset analysis was not found useful in predicting clinical outcome in lupus nephritis at the end of 6 months of intensive treatment.

A new approach to immunosuppressive therapy in patients with glomerulonephritis with nephrotic syndrome

Glomerulonephritis (GN) is a group of immuno-inflammatory kidney diseases with predominant glomerular lesions that are difficult to treat. The greatest problems are caused by the treatment of GN with nephrotic syndrome, which often has a recurrent course. The aim of the research was to study the effectiveness of recombinant interleukin-2 (rIL-2) therapy in the GN patients with nephrotic syndrome. 62 patients with a nephrotic form of primary GN with frequent relapses admitted to the Nephrology Department have been recruited into the study. The age of patients was from 18 to 65 years. The patients underwent standard examinations, as well as immunological studies, before administration of the anti-relapse treatment, and 12 months after the treatment was started. Immunological testing included immunophenotyping of lymphocytes with counting of T and B lymphocytes, immunoregulatory and activated subpopulations of T lymphocytes, determination of urinary immunoglobulins (IgM, IgG, IgA) by immunoturbidimetric assays, proinflammatory cytokines – IL-1β, IL-8, IL-17A and anti-inflammatory cytokine IL-10 by ELISA tests. As a result of studies, the examined patients showed an increased contents of T helper cells, activated T lymphocytes (CD8+HLA-DR+CD45+, CD3+CD8brightCD38+) along with decreased numbers of Treg cells and an increased contents of proinflammatory cytokines IL-1β, IL-8, IL-17A and immunoglobulins of all three classes in urinary samples.The cohort of patients with GN selected for the study was divided in two groups (the main group and the comparison group). In addition to nephroprotective and steroid therapy, the treatment regimen of patients included rIL-2 in the main group, or cyclophosphamide in the comparison group. Regardless of the method used, the levels of protein, IgG and IL-17A in the urine proved to be decreased relative to the initial values; the contents of B cells and HLA-DR+ cytotoxic T lymphocytes in peripheral blood were found to be decreased. The revealed changes were more pronounced in the main group of patients. By 12 months after starting the treatment, the mentioned indexes began to differ significantly in the main group from those in the comparison group. Serum creatinine levels, numbers of T helper cells and Treg cells, IL-1β levels in urine did not undergo significant changes in the comparison group, whereas a decrease in serum creatinine and urinary IL-1β was registered in the main group of patients, along with decreased number of T helpers and increased numbers of Treg cells. In the main group of patients treated with rIL-2, the average number of relapses per year decreased by 4 times, showing only a 1.2-fold decrease in the comparison group. Hence, the low-dose therapy with rIL-2 may be considered an effective and safe alternative to conventional immunosuppressive therapy and a new option of the targeted treatment of glomerulonephritis with frequent recurrence of nephrotic syndrome.

Persistent lymphocyte reduction and interleukin-6 levels are independently associated with death in patients with COVID-19

To investigate the value of the peripheral blood lymphocyte count (LYM) combined with interleukin-6 (IL-6) in predicting disease severity and prognosis in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia. This was a prospective observational cohort study. A total of 109 patients with SARS-CoV-2 pneumonia who were admitted to Nanjing First Hospital from December 2022 to January 2023 were enrolled. The patients were divided into two groups based on disease severity: severe (46 patients) and critically ill (63 patients). The clinical data of all patients were collected. The clinical characteristics, sequential organ failure assessment (SOFA) score, peripheral blood lymphocyte count, IL-6 level and other laboratory test results were compared between the two groups. A receiver operating characteristic (ROC) curve was plotted to evaluate the predictive value of each index for SARS-CoV-2 pneumonia severity; patients were regrouped using the optimal cut-off value of the ROC curve, and the relationship between different LYM and IL-6 levels and the prognosis of patients was analysed. Kaplan‒Meier survival curve analysis was performed; in the different LYM and IL-6 groups, the patients were regrouped based on whether thymosin was used, and the effect of thymosin on patient prognosis was compared between the groups. Patients in the critically ill group were significantly older than patients in the severe group (age: 78 ± 8 vs. 71 ± 17, t = 2.982, P < 0.05), and the proportion of patients with hypertension, diabetes and cerebrovascular disease was significantly higher in the critically ill group than in the severe group (69.8% vs. 45.7%, 38.1% vs. 17.4%, 36.5% vs. 13.0%; χ2 values, 6.462, 5.495, 7.496, respectively, all P < 0.05). Compared with the severe group, the critically ill group had a higher SOFA score on admission (score: 5.4 ± 3.0 vs. 1.9 ± 1.5, t = 24.269, P < 0.05); IL-6 and procalcitonin (PCT) in the critically ill group were significantly higher than those in the severe group on the first day of admission [288.4 (191.4, 412.9) vs. 513.0 (288.2, 857.4), 0.4 (0.1, 3.2) vs. 0.1 (0.05, 0.2); Z values, 4.000, 4.456, both P < 0.05]. The lymphocyte count continued to decline, and the lymphocyte count on the 5th day (LYM-5d) was still low (0.6 ± 0.4 vs. 1.0 ± 0.4, t = 4.515, both P < 0.05), with statistically significant differences between the two groups. ROC curve analysis indicated that LYM-5d, IL-6 and LYM-5d + IL-6 all had value for predicting SARS-CoV-2 pneumonia severity; the areas under the curve (AUCs) were 0.766, 0.725, and 0.817, respectively, and the 95% confidence intervals (95% CI) were 0.676–0.856, 0.631–0.819, and 0.737–0.897, respectively. The optimal cut-off values for LYM-5d and IL-6 were 0.7 × 109/L and 416.4 pg/ml, respectively. LYM-5d + IL-6 had the greatest value in predicting disease severity, and LYM-5d had higher sensitivity and specificity in predicting SARS-CoV-2 pneumonia severity. Regrouping was performed based on the optimal cut-off values for LYM-5d and IL-6. Comparing the IL-6 ≥ 416.4 pg/ml and LYM-5d < 0.7 × 109/L group with the other group, i.e., patients in the non–low-LYM-5d and high-IL-6 group, patients in the low-LYM-5d and high-IL-6 group had a higher 28-day mortality rate (71.9% vs. 29.9%, χ2 value 16.352, P < 0.05) and a longer hospital stay, intensive care unit (ICU) stay and mechanical ventilation time (days: 13.7 ± 6.3 vs. 8.4 ± 4.3, 9.0 (7.0, 11.5) vs. 7.5 (4.0, 9.5), 8.0 (6.0, 10.0) vs. 6.0 (3.3, 8.5); t/Z values, 11.657, 2.113, 2.553, respectively, all P < 0.05), as well as a higher incidence of secondary bacterial infection during the disease course (75.0% vs. 41.6%, χ2 value 10.120, P < 0.05). Kaplan‒Meier survival analysis indicated that the median survival time of patients in the low LYM-5d and high-IL-6 group was significantly shorter than that of patients in the non-low LYM-5d and high-IL-6 group (14.5 ± 1.8 d vs. 22.2 ± 1.1 d, Z value 18.086, P < 0.05). There was no significant difference in the curative effect between the thymosin group and the nonthymosin group. LYM and IL-6 levels are closely related to SARS-CoV-2 pneumonia severity. The prognosis for patients with IL-6 ≥ 416.4 pg/ml at admission and a lymphocyte count < 0.7 × 10 9/L on the 5th day is poor.

Dynamics of the innate immune response profile in patients with coronary heart disease at different terms after coronary artery stenting

Atherosclerosis is accompanied by damage to the vascular endothelium of arteries followed by development of inflammatory response and formation of atherosclerotic plaques. Innate immunity is an important component of this response being the earliest non-specific key mechanism. Our objective was to perform a comprehensive assessment of the cellular link of innate immunity, and to compare the results obtained at various terms after coronary stenting.The study involved 50 patients with coronary atherosclerosis (Group 1), who had clinical indications for stenting of coronary arteries, and 20 volunteers (Group 2), who have no signs of coronary artery disease. The study of immune parameters was carried out before surgery, at 4-5, 9-10 and 28-30 days after operation (during early postoperative period), as well as 6 and 12 months after stenting, i.e. over the late post-surgical period. Phenotyping of peripheral blood monocytes and lymphocytes was performed by flow cytometry using monoclonal antibodies (Beckman Coulter, USA). Intracellular content of Granzyme B was carried out with an FC500 flow laser cytofluorimeter. Metabolic activity of neutrophils was assessed by the NBT test. Alpha defensin was determined in blood plasma by ELISA technique (Hycult Biotech, USA). Statistical analysis was performed using the Statistica 12.0 program (StatSoft, USA). Statistical significance was considered significant at p ≤ 0.05.The numbers of natural killer cells and their activity, as well as those of monocytes, were increased in patients with coronary atherosclerosis. We have also shown a suppression of antigen presentation processes, an imbalance in microbicidal activity of neutrophils, with predominant secretion of antimicrobial peptides. Over the early post-surgical period, significant changes included only decreased content of intracellular Granzyme B on days 4-5, and expression of TLR4 and HLA-DR on days 4-5 and 9-10. During the late period, the patients with coronary artery disease exhibited a significant decrease in the content of some lymphocyte subsets: CD3+CD16+, CD16+Gr+ as well as amounts of monocytes: CD14+CD282+, CD14+CD284+, CD14+CD289+, along with HBT-test activity and α-defensin contents, and increased numbers of HLA-DR-expressing monocytes.There are changes in cellular component of innate immunity, indicating persistent inflammation in patients with coronary heart disease. The dynamics of revealed changes following coronary artery stenting may reflect a lability of assessed indicators mostly over the late postoperative period, thus serving a basis for predicting the outcome of coronary stenting.

T-lymphocytes of the 2nd type of the immune response and their role in enhancing inflammation during the professional activities of firefighters

T lymphocytes of the 2nd type of immune response contribute to the development and exacerbation of inflammation, mainly allergic. Increased inflammation with prolonged exposure to adverse factors during the work of firefighters can lead to the development of various diseases. Evaluation of the immunity of firefighters is important for the appointment of adequate treatment and prevention of infectious and allergic diseases. This paper aimed to analyze the indicators of immunity in employees of the state fire service of EMERCOM of Russia, depending on the age and intensity of the professional workload. The surveyed were men (n = 79), mean age 31 years, work experience from 1 to 22 years, with different workload intensity. In peripheral blood, flow cytometry (Navios, FC 500, Beckman Coulter) was used to evaluate subpopulations of monocytes, the relative number of T lymphocytes of the 2nd type of immune response CD3+CD294+. The concentration of total immunoglobulin E (Immulite) was determined. From nasal secretions, the content of secretory immunoglobulin A (Vector Best) was evaluated (n = 30). Statistical processing of the results was performed using the Statistica 12.0 package (StatSoft). An increase in the number of CD3+CD294+ cells was observed in 16.5%. A direct correlation was found between the number of T lymphocytes 2 and the age of the examined persons (р &lt; 0.05). In the group of firefighters with a more intense workload, an increase in the number of CD3+CD294+ cells were 5 times higher (р &lt; 0.05). Among patients who had any disease of the respiratory tract, an increase in this population was observed statistically significantly more often – in 26% of cases versus 11.5%. A strong direct correlation was found between the number of T lymphocytes 2 and the duration of smoking experience (р &lt; 0.05). A direct correlation was established between the number of T lymphocytes 2 and the concentration of total IgE (р &lt; 0.05). A decrease in secretory IgA in the secret from the nasal passages was observed in 23% of firefighters, in 13% of the examined, the indicator went beyond the upper limit of the reference interval. Significantly more often revealed the deviation of this indicator from the reference values in firefighters with a high workload. An increase in the subpopulation of classical monocytes was established in the group of those examined with a high number of CD3+CD294+ cells (p &lt; 0.05). Thus, with an increase in the professional workload of firefighters in unfavorable conditions of service, inhibition of protection to infection and aggravation of damage to the respiratory tract with an increase in the 2nd type of immune response are noted. Evaluation of the number of type 2 T lymphocytes in peripheral blood will reveal a predisposition to the T2 profile of immune inflammation, which will contribute to a personalized approach to patient management.