Profile Of Peripheral Blood Lymphocytes Research Articles

Aspirin Challenge-Induced Genome-Wide DNA Methylation Profile of Peripheral Blood Lymphocytes in Aspirin-Exacerbated Respiratory Disease.

Genetic variation and epigenetic factors are thought to contribute to the development of hypersensitivity to aspirin. DNA methylation fluctuates dynamically throughout the day. To discover new CpG methylation in lymphocytes associated with aspirin-exacerbated respiratory disease (AERD), we evaluated changes in global CpG methylation profiles from before to after an oral aspirin challenge in patients with AERD and aspirin-tolerant asthma (ATA). Whole-genome CpG methylation levels of peripheral blood mononuclear cells were quantified with an Illumina 860K Infinium Methylation EPIC BeadChip array and then adjusted for inferred lymphocyte fraction (ILF) with GLINT and Tensor Composition Analysis. Among the 866,091 CpGs in the array, differentially methylated CpGs (DMCs) were found in 6 CpGs in samples from all 12 patients with asthma included in the study (AERD, n = 6; ATA, n = 6). DMCs were found in 3 CpGs in the 6 ATA samples and in 615 CpGs in the 6 AERD samples. A total of 663 DMCs in 415 genes and 214 intergenic regions differed significantly in the AERD compared with the ATA. In promoters, 126 CpG loci were predicted to bind to 38 transcription factors (TFs), many of which were factors already known to be involved in the pathogenesis of asthma and immune responses. In conclusion, we identified 615 new CpGs methylated in peripheral blood lymphocytes by oral aspirin challenge in AERD but not in ATA. These findings indicate that oral aspirin challenge induces epigenetic changes in ILFs, specifically in AERD patients, possibly via changes in TF binding, which may have epigenetic effects on the development of AERD.

Aspirin Challenge–Induced Genome-Wide DNA Methylation Profile of Peripheral Blood Lymphocytes in Aspirin-Exacerbated Respiratory Disease

Aspirin Challenge–Induced Genome-Wide DNA Methylation Profile of Peripheral Blood Lymphocytes in Aspirin-Exacerbated Respiratory Disease

Anticentromere antibody positive patients with primary Sjögren's syndrome have distinctive clinical and immunological characteristics.

To investigate the clinical manifestations, immunological characteristics, circulating lymphocyte subsets and risk factors of anticentromere antibody (ACA) positive patients with primary Sjögren's syndrome (pSS). Data of 333 patients with newly diagnosed pSS were collected and analysed retrospectively. The demographic features, glandular dysfunction, extraglandular manifestations, laboratory data, peripheral blood lymphocyte profiles and serum cytokines were compared between ACA-positive and ACA-negative pSS patients. Logistic regression analysis was used to evaluate the association between ACA and pSS characteristics. The prevalence of ACA among pSS patients was 13.5%. ACA-positive pSS patients were older at diagnosis and had longer disease duration. Xerostomia, xerophthalmia, parotid enlargement, Raynaud's phenomenon (RP), lung and digestive system involvement were more common in ACA-positive group, whereas haematological involvement such as leukopenia was more common in the ACA-negative group. Less frequency of rheumatoid factor, hypergammaglobulinaemia, anti-SSA and anti-SSB positivity, as well as higher positivity rate of ANA were observed in ACA-positive pSS patients, who exhibited a lower ESSDAI. In addition, decreased B cells and elevated NK cells were found in ACA-positive patients. Multivariate analysis identified that disease duration longer than 5 years, parotid enlargement, normal immunoglobulin and the absence of anti-SSA antibody were risk factors of ACA-positive pSS. ACA positive pSS patients have distinctive clinical manifestations and less severe immunological features, present a lower disease activity and lower activation of the humoral immune system. Physicians should pay attention to RP, lung and liver involvement in this subset of pSS.

Subpopulation composition of peripheral blood lymphocytes in COVID-19

The pathogenesis of severe coronavirus infection COVID-19 is associated with activation of immune system, cytokine storm, impaired blood clotting, microvascular thrombosis, organ ischemia and multiple organ dysfunction syndrome. The role of various lymphocyte subpopulations in COVID-19 is still debated. The aim of our study was to analyze the subpopulational profile of peripheral blood lymphocytes in COVID-19 patients as compared with healthy donors.The study included 20 COVID-19 patients (11 males and 9 females,) and 26 healthy donors. Average age of the patients was 52 and 56 years, respectively. Clinical examinations were performed by standard laboratory methods. Peripheral blood lymphocytes were isolated in the Ficoll gradient. The cells were stained with antibodies to specific antigens of main lymphocyte populations, endothelial cells, and apoptotic cell markers. The analysis was performed by flow cytometry. The results showed that all patients had elevated C-reactive protein (14- to 35-fold), ferritin (1.2- to 13-fold), D-dimers (1.2- to 90-fold). 55% of men had a decrease in the absolute number of lymphocytes, in women this index was at the low normal limit. Cytometric analysis showed that, among peripheral blood lymphocytes, the proportion of functional cells expressing the CD45 marker ranged from 2 to 12% in 70% of patients, as compared with 80-99% among the donors. The proportion of CD45+ lymphocytes significantly correlated with the level of hemoglobin, but not with the levels of inflammatory biochemical markers. Among the functional lymphocytes of patients, there was a decrease in the proportion of CD3+, CD4+, CD8+T cells, increased proportion of natural killer CD56+ and the apoptotic (AnnexinV+) cell contents, but the proportion of CD19 and HLA-DR+B cells was not changed. Analysis of the lymphocyte (LC) subpopulations that did not express CD45 marker showed that this fraction contained different lymphocyte subsets with reduced expression of CD4, CD8, CD19, CD56 etc. in the blood of patients and donors. Higher percentage of endothelial cells expressing CD62P marker made the difference between patients and donors.Laboratory determination of lymphocyte subsets in blood samples of COVID-19 patients does not reflect the real severity pattern of the disease, thus requiring studies of the CD45-expressing functional cell populations.

Genome-wide DNA methylation profile of peripheral blood lymphocytes from subjects with nonsteroidal anti-inflammatory drug-induced respiratory diseases.

Significant changes in CpG methylation have been identified in nasal polyps, which are the main targets of nonsteroidal anti-inflammatory drug-exacerbated respiratory disease (NERD); however, these polyps are composed of various cellular components. In the present study, whole-genome CpG methylation in peripheral blood lymphocytes (PBLs) was analyzed to define the epigenetic changes in lymphocytes, which are the primary immune cells involved in NERD. Genomic DNA from peripheral blood mononuclear cells from 27 NERD and 24 aspirin-tolerant asthma (ATA) was subjected to bisulfate conversion and a methylation array. Quantitative CpG methylation, the β-values as a quantitative measure of DNA methylation, in lymphocytes were calculated after adjustments for cellular composition. Fifty-six hypermethylated and three hypomethylated differentially methylated CpGs (DMCs) in PBLs in the NERD compared with ATA. The top 10 CpG loci predicted the methylation risk score, with a positive predictive value of 91.3%, a negative predictive value of 81.5% and an accuracy of 84.3%. As demonstrated in the nasal polyps, 30 DMCs were predicted to bind to the following 10 transcription factors, ranked in descending order: AP-2alphaA, TFII-1, STAT4, FOXP3, GR, c-Est-1, E2F-1, XBP1, ENKTF-1 and NF-1. Gene ontology analysis identified 13 categories such as regulation of T-helper 17 cell differentiation, including SMAD7 and NFKBIZ. PBLs in NERD contained no DMCs in genes associated with the prostaglandin and leukotriene pathways, which were found in ATA. PBLs in NERD form a unique pattern of DNA CpG methylation, and the combined analysis may provide predictive values for NERD.

Effect of dietary AGEs on the transcriptional profile of peripheral blood lymphocytes

BackgroundAdvanced Glycation End Products (AGEs) are formed in foods subjected to high temperatures. Although endogenous AGEs are implicated in certain autoimmune diseases, the role of dietary AGEs remains to be investigated. Materials and methodsSamosa (Indian snack) samples in fried and baked form were collected from street food vendors. AGEs were extracted from them and quantified using fluorescence spectrophotometry. Peripheral blood lymphocytes (PBLs) were incubated with AGEs to study the effect on their cell cycle and transcriptional expression of Foxp3, RORγT, IL-17, IL-10, p21. ResultThe AGE-specific fluorescence of fried samosa was significantly (p < 0.05) higher than the baked one. PBLs showed significantly reduced Foxp3, IL-10, and p21 but no significant change in RORγT and IL-17 expression on incubation with AGEs. ConclusionExposure to dAGEs may result in perturbation of balance between T-cell suppressive and activating factors that may play a role in driving PBLs towards an autoimmune phenotype. Suitable modifications in dietary habits to reduce them may be a valuable strategy to fight autoimmunity.

Role of apoptosis disturbances in external genital endometriosis

External genital endometriosis (EGE) is currently considered one of the most common disorders in women at their reproductive age. EGE is a chronic disease that affects about 10% of women of reproductive age, leading to functional and structural changes in reproductive system, infertility, pelvic pain, and affects their quality of life. Moreover, the patients with endometriosis often suffer from depression, especially, due to pain syndrome. Endometriosis is associated with dysregulation of T-cell immune response, which presents as altered interactions between T-cells, macrophages, NK (natural killer cells), B-cells, thus allowing ectopic implantation of endometrial cells. The pronounced negative effect of EGE on the quality of life of patients, their reproductive function, impaired adaptation of the patients in community, and growing costs of treatment determine social significance of the disease and importance of appropriate studies. It is obvious that disorders of the immune system play a significant role in IGE development. However, despite extensive research in this area, the processes of apoptosis in IGE have not been sufficiently studied. Therefore, the aim of our study was to assess the origin of apoptosis disorders in women with stage I-II and III-IV of external genital endometriosis. We examined 71 patients with EGE which were divided into 2 groups: group 1 included the patients with EGE stage I-II (n = 31), and group 2, the patients with stage III-IV EGE (n = 40). The control group included 24 patients without EGE. The population profile of peripheral blood lymphocytes was determined by laser flow cytometry using Immunotex reagents, i.e., FITC-labeled CD8+, CD16+, and PE-labeled CD95+ antibodies. The number of lymphocytes entering apoptosis was detected using a diagnostic kit for AnnexinV+ (FITC) and PI+ from Caltag. The results were read on a BECKMAN COULTER EPICS XL-II flow cytometer using standard protocols.An imbalance between activation and apoptosis of immunocompetent cells may be a significant cause of EGE. Induction of apoptosis by the Fas-FasL pathway in patients with external genital endometriosis leads to decreased cytotoxicity mediated by T-lymphocytes and NK-cells. A statistically significant decrease in the content of AnnexinV+ presenting lymphocytes suggests impairment of their receptor-dependent apoptosis.Altered apoptosis processes may be responsible for the development of IGE by promotion of proliferation and growth of endometrioid implants.

Peripheral blood lymphocyte phenotypes in Alzheimer and Parkinson’s diseases

IntroductionNeuroinflammation is involved in the pathophysiology of various neurological disorders, in particular Alzheimer disease (AD) and Parkinson’s disease (PD). Alterations in the blood-brain barrier may allow peripheral blood lymphocytes to enter the central nervous system; these may participate in disease pathogenesis. ObjectiveTo evaluate the peripheral blood lymphocyte profiles of patients with AD and PD and their association with the disease and its progression. MethodsThe study included 20 patients with AD, 20 with PD, and a group of healthy individuals. Ten of the patients with AD and 12 of those with PD were evaluated a second time 17 to 27 months after the start of the study. Lymphocyte subpopulations and their activation status were determined by flow cytometry. All patients underwent neurological examinations using internationally validated scales. ResultsCompared to healthy individuals, patients with AD and PD showed significantly higher levels of activated lymphocytes, lymphocytes susceptible to apoptosis, central memory T cells, and regulatory T and B cells. As the diseases progressed, there was a significant decrease in activated cells (CD4+ CD38+ and CD8+ CD38+ in PD and AD, CD4+ CD69+ and CD8+ CD69+ in PD), T cells susceptible to apoptosis, and some regulatory populations (CD19+ CD5+ IL10+ in PD and AD, CD19+ CD5+ IL10+ FoxP3+, CD4+ FoxP3+ CD25+ CD45RO+ in PD). In patients with AD, disease progression was associated with lower percentages of CD4+ CD38+ cells and higher percentages of effector CD4 cells at the beginning of the study. Significant differences were observed between both diseases. ConclusionsThis study provides evidence of changes in peripheral blood lymphocyte phenotypes associated with AD and PD and their severity. Considering effective blood-brain communication, our results open new avenues of research into immunomodulation therapies to treat these diseases.

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.

Malignant ascite-derived extracellular vesicles inhibit T cell activity by upregulating Siglec-10 expression.

Background and purpose: To evade immune defense, cancer cells can employ extracellular vesicles (EVs) to inhibit the anti-tumor activity of lymphocytes in the tumor microenvironment. However, the mechanisms and key molecules that mediate the effects of EVs on lymphocytes are unclear.Patients and methods: We used Quantibody® Human Cytokine Antibody Array 440 to determine the tumor immunity-related cytokine profile of peripheral blood lymphocytes (PBLs) stimulated with EVs derived from peritoneal washes or malignant ascites. We detected 21 upregulated and 27 downregulated proteins, including the immunosuppressive receptors Siglec-10, SLAM, PD-1, and TIM-3.Results: Flow cytometry analysis of PBLs or ovarian cancer ascites suggested that Siglec-10 expression on CD3+ T cells was higher in ovarian cancer patients than in healthy controls and in the malignant ascites of ovarian cancer patients than in their blood. Moreover, the expression of CD24, the Siglec-10 ligand, was associated with tumor stage and cancer cell metastasis. Finally, compared to the benign peritoneal wash-derived EVs, the malignant EVs significantly upregulated Siglec-10 expression on Jurkat T cells, inhibited the protein kinase C activity induced by phorbol 12-myristate 13-acetate and ionomycin, and impaired the phosphorylation of the tyrosine kinase ZAP-70 activated by crosslinking with an anti-CD3 antibody.Conclusion: The EVs secreted by malignant ovarian cells upregulated Siglec-10 expression on T cells and impaired T cell activation in the tumor microenvironment. We believe that a comprehensive understanding of the regulation of Siglec-10 and CD24 by malignant EVs has clinical importance, as it will aid in the development of better immunotherapeutic strategies for ovarian cancer.

Fenotipos de linfocitos periféricos en las enfermedades de Alzheimer y Parkinson

IntroductionNeuroinflammation is involved in the pathophysiology of various neurological disorders, in particular Alzheimer disease (AD) and Parkinson's disease (PD). Alterations in the blood-brain barrier may allow peripheral blood lymphocytes to enter the central nervous system; these may participate in disease pathogenesis. ObjectiveTo evaluate the peripheral blood lymphocyte profiles of patients with AD and PD and their association with the disease and its progression. MethodsThe study included 20 patients with AD, 20 with PD, and a group of healthy individuals. Ten of the patients with AD and 12 of those with PD were evaluated a second time 17 to 27 months after the start of the study. Lymphocyte subpopulations and their activation status were determined by flow cytometry. All patients underwent neurological examinations using internationally validated scales. ResultsCompared to healthy individuals, patients with AD and PD showed significantly higher levels of activated lymphocytes, lymphocytes susceptible to apoptosis, central memory T cells, and regulatory T and B cells. As the diseases progressed, there was a significant decrease in activated cells (CD4+ CD38+ and CD8+ CD38 + in PD and AD, CD4+ CD69+ and CD8+ CD69+ in PD), T cells susceptible to apoptosis, and some regulatory populations (CD19+ CD5+ IL10+ in PD and AD, CD19+ CD5+ IL10+ FoxP3+, CD4+ FoxP3+ CD25+ CD45RO+ in PD). In patients with AD, disease progression was associated with lower percentages of CD4+ CD38+ cells and higher percentages of effector CD4 cells at the beginning of the study. Significant differences were observed between both diseases. ConclusionsThis study provides evidence of changes in peripheral blood lymphocyte phenotypes associated with AD and PD and their severity. Considering effective blood-brain communication, our results open new avenues of research into immunomodulation therapies to treat these diseases.

Depression of lymphocyte activity during cutaneous leishmaniasis: a case report

Skin leishmaniasis includes lesions of different appearance, shape, and severity, spanning from alarming diffuse lesions to an asymptomatic course. Moreover, aspecific presentation, as well as challenging differential diagnosis of cutaneous leishmaniasis, may request more in-depth investigations on the intriguing and complex pathogenesis of such infection. A 7-year case of worsening cutaneous leishmaniasis in the left frontoparietal region of the scalp, achieving omolateral eyebrow, in a 68-year-old male patient prompted us to address the immunity profile of peripheral blood lymphocytes. An increase of regulatory CD19+/CD38bright/CD24bright B cell lymphocytes was observed at the front of normal levels of other lymphocytes subpopulations, including CD4+/CD25bright T cells.The total IgG and IgM, as well as proinflammatory subclasses of IgG, were below the normal range. However, IgG4 subclass was found normal. In conclusion, our data may indicate inhibition of humoral immunity associated with an increase of lymphocyte B-regulatory subpopulation.

Mucosal immune responses to b-lactoglobuin (βLG)-immunized mice are altered following colitis challenge

Abstract Food allergens induce mucosal immune responses, and their continual exposure to sensitive hosts leads to health problems, e.g. intestinal inflammation. We tested dextran sodium sulfate (DSS) to disrupt intestinal homeostasis in mice immunized with βLG. BALB/c mice were ip. sensitised with βLG/PBS, thereafter orally challenged with βLG. DSS was delivered in drinking water et libitum. Humoral response and T cell profile were monitored for differences in immune response between groups. The highest level of Ag-specific serum IgG was observed in the i.p. immunized group without dependence on DSS presence. The level of Ag-specific IgA in sera and fecal extracts was low in each group. We found the greatest amount of CD4+ and FoxP3+CD25+CD4+ T cells in the spleens, mesenteric and peripheral lymph nodes obtained from mice orally immunized with βLG and receiving DSS treatment. DSS also induced a subset of CD4+ T cells producing a high amount of IFN-γ in all experimental groups. An elevated number IFN-γ+ CD4+ T cells was found in the lymphoid tissues of βLG-immunized mice (ip and orally) and treated with DSS. The profile of peripheral blood lymphocytes showed a two-fold increase in FoxP3+CD25+CD4+ T cells after DSS stimulation. The proposed experimental model of the inflamed intestine provoked with allergens showed differences in humoral responses and lymphocyte profiles, demonstrating that this regimen can be used to monitor immunoreactivity to food compounds.

The state of immune system during the use of probiotic lactobacilli in complex treatment of papillomavirus infection

Aim. Assessment of impact of probiotic lactobacilli complex on treatment efficiency and normalization of immune status in women with human papillomavirus infection (PVI).Methods. Total of 65 patients of reproductive age with cervical pathology and PVI were examined. «Provag» was included into the standard therapy as the source of probiotic lactobacilli complex. Immunological studies were conducted before and after the treatment. The phenotypic profile of peripheral blood lymphocytes was determined by means of flow cytometry. The concentration of immunoglobulin A, G and M in serum was determined by ELISA.Results. Decreased amounts of T- and NK-cells were observed in PVI patients on day 1. Regardless of treatment method, by day 30 the number of T-lymphocytes in the blood of women with PVI increases. Percentage of NK-cells remained below the normal values on day 30 in the group of patients who received traditional scheme of PVI treatment. Patients with PVI taking additionally probiotic lactobacilli complex showed an increase of NK-cells by the end of the treatment. On day 1 decrease of relative and absolute numbers of T-helper cells was detected. Regardless of the method of treatment by day 30 the number of T-helper cells increases to the reference level. IgM and IgG levels in peripheral blood of women with PVI were increased even on day 1. By the end of the observation period their concentration in patients with conventional treatment scheme remained increased. Patients receiving the standard therapy supplemented with «Provag» demonstrated the decrease of these classes of immunoglobulins to the reference level.Conclusion. As a result of treatment normalization of NK-cells and T-helpers numbers was observed whereas the IgG and IgM levels remained increased.

Mir Expression Profile of Peripheral Blood Lymphocytes Predicts Relapse in Pediatric Acute Lymphoblastic Leukemia

Abstract Introduction: Our previous findings showed that the microRNA (miR) expression profile of miR-151, miR-1290 and miR-451 measured in bone marrow (BM) aspirations taken at diagnosis can predict risk of relapse in pediatric acute lymphoblastic leukemia (ALL) patients treated according to BFM and DCOG protocols (Avigad et al., Genes, Chromosomes &amp; Cancer; 55:328-339, 2016). The development of a minimally invasive blood test for predicting relapse in pediatric B-ALL patients can greatly benefit patients. Therefore, the aim of this current retrospective exploratory study was to investigate the predictive ability of miR-151-5p, miR-1290 and miR-451 measured in peripheral blood lymphocytes (PBL) at diagnosis. Methods: BM (n=67) and PBL (n=22) samples were collected at Schneider Children's Medical Center of Israel between 1995 to 2008. Twenty cases had paired BM and PBL samples. The expression levels of miR-151-5p, miR-1290 and miR-451 were measured in both BM and PBL samples from B-ALL patients (mean age = 6.6 years, median 5.5 years, range 1.3 to 19 years) taken at diagnosis. Pearson correlation (n=20) was applied for assessing the correlation between BM and PBL following logarithmic transformation. Kaplan Meier analysis was performed for testing relapse free survival (RFS). The cutoff value used for survival analysis for each miR, was determined by the highest Youden index (J=Sensitivity + Specificity - 1). Results: Similar to our previous study all three miRs measured in BM samples (n=67) were able to predict relapse risk (p=0.027). The expression levels measured in BM and PBL samples of all three miRs were significantly correlated (miR-151-5p, r=0.764, p&lt;0.0001; miR-1290, r=0.725, p&lt;0.0001; miR-451, r=0.553, p=0.011). For Kaplan Meier analysis, we defined "positive" as equal or below the cutoff for miR-151-5p or miR-451 and above the cutoff for miR-1290. In PBL samples, only miR-151-5p (p=0.002) and miR-1290 (p=0.004) maintained their prognostic significance. When combining those two miRs, patients who had positive expression levels in PBL of at least one miR had significantly lower RFS of 25% vs. 100% in the negative patients (p=0.0002, Figure 1A). Similarly, the paired BM samples maintained significant inferior RFS of 43% vs. 92% in the negative patients (p=0.011, Figure 1B). Conclusions: Expression levels of miR-151 and miR-1290 in PBL are useful for predicting relapse risk. Results from this exploratory investigation established the feasibility for continuing research and performing a larger prospective clinical trial using blood samples. Moreover, we will investigate the utility of the miRs for long-term monitoring of B-ALL patients during follow-up. The development of a blood test based on miR-151 and miR-1290 for monitoring B-ALL patients risk for relapse will be pursued in parallel to the development of the bone marrow test. These findings should be interpreted with caution due to the small cohort size. Partly supported by Curewize Health Ltd. Figure 1. Figure 1. Disclosures Avigad: Curewize Health: Consultancy, Patents &amp; Royalties: Patent Applicant of Curewize IP. Shichrur:Curewize Health: Employment, Patents &amp; Royalties: Patent Applicant of Curewize IP. Dotan:Curewize Health: Employment, Other: Options Holder in Curewize Health. Yarden:Curewize Health: Employment, Equity Ownership. Yaniv:Curewize Health: Consultancy, Patents &amp; Royalties: Patent Applicant of Curewize IP.

Changes of the Immunophenotypic Spectrum and the Enzymatic Profile of Peripheral Blood Lymphocytes in Infants with Hypertrophy of the Pharyngeal Tonsil

to study immunophenotype and NAD- and NAD(P)-dependent dehydrogenase of blood lymphocytes activity indicators in children with hypertrophy of the pharyngeal tonsils (HPT). 57 children aged 1-3 years with HPT were examined. The focus group included 35 healthy children of the similar age. The number of CD₃⁺-, CD₄⁺-, CD₈⁺-, CD₁₆⁺/₅₆⁺-, CD₁₉⁺-cells in the blood was determined by flow cytometry. The activity of NAD(P)-dependent dehydrogenase was studied by the method of A. Savchenko and coauth. (1989). The changes of immunophenotypic spectrum of peripheral blood lymphocytes in infants with HPT have been revealed. The increase of ribose-5-phosphate and NADN-dependent reactions of macromolecular synthesis, the reduction of malataspartat shunt role in cell energy, the reduction of anaerobic lactate dehydrogenase reaction, the compensatory increase in the glycerol-3-phosphate dehydrogenase activity, the high substrate flow of the citric acid cycle and the reduced level of glutathione have been fixed. The correlation analysis has showed increase in the number of correlations between indicators of investigated oxidoreductase activity in blood lymphocytes in children with HPT and the high level of correlation between the metabolic reactions of the mitochondrial compartment. the change of immunophenotype, enzymatic activity, correlation pattern of connection between intracellular enzymes of peripheral blood lymphocytes have been revealed in children aged 1-3 years with HPT.

Cross-reactivity of commercially available anti-human monoclonal antibodies with canine cytokines: establishment of a reliable panel to detect the functional profile of peripheral blood lymphocytes by intracytoplasmic staining.

BackgroundThe process for obtaining monoclonal antibodies against a specific antigen is very laborious, involves sophisticated technologies and it is not available in most research laboratories. Considering that most cytokines remain partially conserved among species during evolution, the search for antibody cross-reactivity is an important strategy for immunological studies in veterinary medicine. In this context, the amino acid sequence from human and canine cytokines have demonstrated 49–96 % homology, suggesting high probability of cross-reactivity amongst monoclonal antibodies. For this, 17 commercially available anti-human monoclonal antibodies [IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-10, IL-12, IL-13, IL-17A, IFN-γ (#1, #2), TNF-α (#1, #2) and TGF-β], were evaluated in vitro for intracellular cytokine detection in a stimulated canine blood culture by flow cytometry and confocal microscopy. Lymphocytes from peripheral blood of healthy and two unhealthy dogs were analyzed.ResultsEleven anti-human mAbs [IL-1α, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-12, IL-17A, TNF-α (#1, #2) and TGF-β] cross-reacted against canine intracellular cytokines. The specificity of the assays was not affected after Fc-blocking. Three anti-human cytokine mAbs [IL-4, IL-8 (#2) and TGF-β] when evaluated by confocal microscopy also cross-reacted with intracellular canine cytokines. The identification of human mAbs that cross-reacted with canine cytokines may support their use as immunological biomarkers in veterinary medicine studies.ConclusionThe identification of these 11 anti-human cytokine mAbs that cross-reacted with canine cytokines will be useful immunological biomarkers for pathological conditions by flow cytometry and fluorescence microscopy in dogs.

Interleukin-15 is required for immunosurveillance and immunoprevention of HER2/neu-driven mammary carcinogenesis.

IntroductionWe previously demonstrated that HER2/neu-driven mammary carcinogenesis can be prevented by an interleukin-12 (IL-12)-adjuvanted allogeneic HER2/neu-expressing cell vaccine. Since IL-12 can induce the release of interleukin-15 (IL-15), in the present study we investigated the role played by IL-15 in HER2/neu driven mammary carcinogenesis and in its immunoprevention.MethodsHER2/neu transgenic mice with homozygous knockout of IL-15 (here referred to as IL15KO/NeuT mice) were compared to IL-15 wild-type HER2/neu transgenic mice (NeuT) regarding mammary carcinogenesis, profile of peripheral blood lymphocytes and splenocytes and humoral and cellular responses induced by the vaccine.ResultsIL15KO/NeuT mice showed a significantly earlier mammary cancer onset than NeuT mice, with median latency times of 16 and 20 weeks respectively, suggesting a role for IL-15 in cancer immunosurveillance. Natural killer (NK) and CD8+ lymphocytes were significantly lower in IL15KO/NeuT mice compared to mice with wild-type IL-15. The IL-12-adjuvanted allogeneic HER2/neu-expressing cell vaccine was still able to delay mammary cancer onset but efficacy in IL-15-lacking mice vanished earlier: all vaccinated IL15KO/NeuT mice developed tumors within 80 weeks of age (median latency of 53 weeks), whereas more than 70 % of vaccinated NeuT mice remained tumor-free up to 80 weeks of age. Vaccinated IL15KO/NeuT mice showed less necrotic tumors with fewer CD3+ lymphocyes and lacked perforin-positive infiltrating cells compared to NeuT mice. Concerning the anti-vaccine antibody response, antibody titer was unaffected by the lack of IL-15, but less antibodies of IgM and IgG1 isotypes were found in IL15KO/NeuT mice. A lower induction by vaccine of systemic interferon-gamma (IFN-γ) and interleukin-5 (IL-5) was also observed in IL15KO/NeuT mice when compared to NeuT mice. Finally, we found a lower level of CD8+ memory cells in the peripheral blood of vaccinated IL15KO/NeuT mice compared to NeuT mice.ConclusionsWe demonstrated that IL-15 has a role in mammary cancer immunosurveillance and that IL-15-regulated NK and CD8+ memory cells play a role in long-lasting immunoprevention, further supporting the potential use of IL-15 as adjuvant in immunological strategies against tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0588-x) contains supplementary material, which is available to authorized users.

Immunological profiling of haemodialysis patients and young healthy individuals with implications for clinical regulatory T cell sorting.

With the increasing interest in clinical trials with regulatory T cells (Tregs), immunological profiling of prospective target groups and standardized procedures for Treg isolation are needed. In this study, flow cytometry was used to assess peripheral blood lymphocyte profiles of young healthy individuals and patients undergoing haemodialysis treatment. Tregs obtained from the former may be used in haematopoietic stem cell transplantation and Tregs from the latter in the prevention of kidney transplant rejection. FOXP3 mRNA expression with accompanying isoform distribution was also assessed by the quantitative reverse transcriptase polymerase chain reaction. Flow-cytometric gating strategies were systematically analysed to optimize the isolation of Tregs. Our findings showed an overall similar immunological profile of both cohorts in spite of great differences in both age and health. Analysis of flow-cytometric gating techniques highlighted the importance of gating for both CD25high and CD127low expression in the isolation of FOXP3-positive cells. This study provides additional insight into the immunological profile of young healthy individuals and uraemic patients as well as in-depth analysis of flow-cytometric gating strategies for Treg isolation, supporting the development of Treg therapy using cells from healthy donors and uraemic patients.

Effects of entecavir on peripheral blood lymphocyte profiles in chronic hepatitis B patients with suboptimal responses to adefovir.

The aim of the present study was to assess the long-term impact of entecavir (ETV) on T, B and natural killer (NK) cell immunity in patients with suboptimal responses to adefovir (SRA) chronic hepatitis B (CHB). Thirty SRA CHB patients and 20 age- and gender-matched healthy controls (HC) completed at least 6 months of ETV treatment. Hepatitis B virus (HBV) DNA loads, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the frequency of different subsets of T, B and NK cells in individual subjects were measured. There were smaller numbers of CD3(-) CD56(+) and CD244(+) NK cells, CD3(+) CD8(+) T cells and cytokine-secreting CD4(+) T cells, but greater numbers of CD3(+) CD4(+) , CD4(+) CD25(+) Foxp3(+) , CD4(+) CD25(+) CD127(low) T cells and CD19(+) CD27(+) B cells, detected in SRA patients. After switching to ETV monotherapy, the levels of HBV DNA and hepatitis B s antigen, as well as hepatitis B e antigen seropositivity, decreased gradually, accompanied by decreases in ALT and AST levels. Furthermore, the number of NK, CD8(+) and cytokine-secreting CD4(+) T cells increased, whereas the number of CD4(+) , CD4(+) CD25(+) Foxp3(+) , CD4(+) CD25(+) CD127(low) T cells and CD19(+) CD27(+) B cells decreased, in SRA CHB patients. The frequency of CD4(+) interferon-γ-positive T cells was negatively associated with serum HBV DNA levels. Thus, treatment with ETV inhibits HBV replication, modulates T and NK cell immunity and improves liver function in SRA CHB patients.