Three-color Flow Cytometry Research Articles

Changes in the immunophenotype of endometrium during implantation window receptivity formation in healthy fertile women

IntroductionEndometrial receptivity is largely determined by the immunophenotype of endometrium, especially uterine NK-cells (uNK). Immune component is directly involved in the formation of favourable microenvironment for the blastocyst implantation and placenta formation, but the way it changes during the maturation of endometrial tissue in healthy fertile women is still underexplored. MethodsThe endometrium was collected from 47 healthy oocyte donors after controlled ovarian stimulation: 23 women on the day of oocytes retrieval (OR) and 24 women on the term of implantation window (IW). The OR group was analysed, published previously and used as a comparison group to show the dynamic of changes.Isolated endometrial lymphocytes and peripheral blood samples were stained with monoclonal antibodies and analysed according to the three-color flow cytometry protocol. ResultsThe proportion of NK-cells (CD3−CD56+) in endometrium grew significantly in the implantation window compared to the oocytes retrieval day. NK-cells acquired a more differentiated phenotype from the day of OR until IW: the expression of CD8 and CD158a significantly increased, while the expression of HLA-DR significantly decreased. Significant correlations between peripheral blood and endometrial NK-cells were found in CD8 expression during OR and IW, CD335(p46)neg and CD335(p46)++ subsets during IW term. DiscussionImmunophenotype of receptive endometrium forms due to the accumulation of uNK-cells, which actively proliferate, become mature, differentiative, and ready to meet the embryo.Endometrial immunophenotype is peculiar and specific but not autonomic and isolated.Differentiation (CD8 on NK-cells), and activity (p46 on NK-cells) of peripheral blood lymphocytes is reflected in endometrial lymphocytes profile, and therefore the research of peripheral blood immunophenotype is relevant.

CD69 flow cytometry to complement interferon-γ release assay for active tuberculosis.

The interferon-γ (IFN-γ) release assay (IGRA) is widely used to diagnose tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb). However, indeterminate IGRA results due to "high Nil" or "low PHA" responses limit its clinical utility. We developed a novel assay using CD69 flow cytometry (FC) to complement IGRA. CD69 FC measures the surface CD69 expression on T cells prior to centrifugation to harvest the plasma for IGRA. T cell responses against Mtb antigen 1 (Ag1) or Ag2 were measured using three-color FC (CD3, CD4, and CD69) in TB (n=140) and non-TB groups (n=117). The cutoff values of Δ%CD69bright cells (stimulated minus unstimulated) for CD4+ and CD4- T cells were established based on healthy individuals (n=63). The assay performances of CD69 FC and IGRA were compared. In subjects with determinate IGRA results ("positive" or "negative"; n=216), the diagnostic accuracies of CD69 FC (90.3%) and IGRA (87.0%) were not significantly different (p=0.31). For indeterminate IGRA results (n=40), CD69 FC attained a diagnostic accuracy of 92.5%. The CD4+ /CD4- ratio within CD69bright T cells measured by CD69 FC was significantly higher (p < 0.05) in the active TB group (6.39 ± 132.05; n=72) than in other CD69 FC-positive subjects (2.84 ± 15.36; n=63) (p < 0.05), whereas CD8 responses expected by IGRA (difference of IFN-γ levels between Mtb Ag tubes) did not differ significantly (0.00 ± 9.18 and 0.00 ± 4.25, respectively, IU/ml; p=0.58). We demonstrated the potential of CD69 FC as a simple, rapid assay for clarifying indeterminate IGRA results and identifying active TB. With further improvements, CD69 FC may complement the IGRA to enhance TB risk stratification in the routine diagnostic workup.

Expression of natural cytotoxicity receptor NKp46 on peripheral blood natural killer cells in women with a history of recurrent implantation failures.

The peripheral blood natural killer (NK) cells diversity is highly complex; recent studies described more than a thousand phenotypes sharing NK cell receptors, across the leukocyte lineages. In this study, we investigated the expression of NKp46 in peripheral blood NK cells in women with a history of recurrent implantation failures (RIF) with euploid embryos with pre-implantation genetic diagnosis (PGD) and control group (donors of oocytes and surrogate mothers). The expression of NKp46 in peripheral blood lymphocytes and NK cells from women with RIF (n = 57) and control group (n = 50) was analyzed with three-color flow cytometry. The percentage of NKp46+ NK cells was significantly higher in women with RIF compare with the control group and high amount of NKp46+ NK cells (>13% of total lymphocytes) was a poor prognostic factor for embryo implantation. Also, women with RIF had a low amount of NKp46neg NK cells, which was a negative prognostic factor for embryo implantation. The analysis of NK subpopulations, on the basis of NKp46 expression, also revealed that NKp46neg NK in low amounts (<20% of NK cells) and NKp46dim in high amounts (>50% of NK cells) are also negative prognostic factors for embryo implantation. Our results support the clinical significance of the NKp46 expression on NK cells in women with RIF. We suggest that the low level of NKp46neg subset in women with RIF may be a result of an imbalance in the differential development of ILC subsets toward cytotoxic ILC (NK cells), which in turn is a negative condition for successful embryo implantation.

Cellular Immune Response (CD8+CD38+) in Relation to Hepatitis B Virus DNA Level and HBsAg Quantification in Hepatitis B Patients

Introduction: Hepatitis B Virus (HBV) infection is a global public health concern. The immune response in HBV represents a key factor in patient outcome. However, the relationship between viral replication and host immune reactivity remains a matter of investigation. Aim: The aim of our study was to investigate whether the cellular immune response of recently diagnosed and treatment naive chronic hepatitis B(CHB) patients may be influenced by the replicative status of HBV. Towards this aim, the correlation between HBV viral load, HBsAg quantification and peripheral T-cell subpopulations CD8+CD38+. Methods: Proportions and absolute counts of CD8 CD38 T cells were determined using three-color flow cytometry in chronic hepatitis B patients (n=50) and healthy controls (n=35). Chronic hepatitis B patients were regularly followed for 48 weeks, during which period the T cell subsets, serum viral load and HBsAg quantitation were measured every 24 months. Results: There was a high level of CD8+CD38+% during the pretreatment stage (Mean 32.4514, standard deviation (SD) 16.8007) compared to the control group (Mean 19.4628, SD 9.75555), p=0.000. Significant decreases in CD8 count were detected 12 months after treatment initiation of HBV therapy (Mean 1359.44, SD 724.362) compared to the control group (Mean 1944.13, SD 948.931), p=0.001. There is a significant correlation between CD8+CD38+ count and serum HBV DNA. A Positive correlation was found between CD8+CD38+ count and HBsAg quantitation. Conclusion: There was a positive correlation between CD8+CD38+ T cells and HBsAg quantitation. The combined use of CD8+CD38+ T cells, HBsAg quantitation and HBV DNA assessment in patients with CHB may guide the clinicians as they guage the likelihood of treatment response.

New early prognostic criteria for pregnancy outcome in women with recurrent miscarriage.

To determine the new criteria for predicting the outcome of pregnancy in women with habitual abortion based on features of differentiation of naive T cells and memory cells in a population of T-helper (CD4+) and cytotoxic T lymphocytes (CD8+). The study involved 61 women with threatened and habitual abortion in the first trimester of gestation. Depending on the outcome of pregnancy was allocated to 3 groups: I went to 39 women whose pregnancy ended in timely delivery; in II - 10 women whose pregnancies ended in premature birth; in III - 11 patients in whom there was a spontaneous miscarriage. Using three-color flow cytometry as peripheral venous blood in populations of CD8+ and CD4+ determined by the content Tn, Tcm, Tem and Temra cells. Statistical analysis was carried out in the program «MicrosoftOffice 2010», «Statistica for Windows 6.0» and MedCalc». When conducting a retrospective assessment, it was found that in the group of patients whose pregnancy ended in preterm delivery, the percentage of CD4+ Tem memory cells was significantly higher and CD4+ Tn lower than in the subgroup with timely delivery (p = 0.013 and p = 0.025, respectively ) In patients with early spontaneous miscarriage, the level of CD8+ Tn significantly decreased against the background of the growth of CD8+ Tem memory cells compared with the same parameters in patients with timely delivery (p = 0.040 and p = 0.014, respectively). Prediction of spontaneous abortion is possible up to CD4+ Tn equal to 34.2% or less (sensitivity - 100.0%, specificity - 56.4%, accuracy - 63.8%), premature birth - if the CD4+ Tn equal to 35, 2% or less (sensitivity - 66.7%, specificity - 74.4% accuracy - 72.9%). Thus, the new criteria will allow additional time to identify risk and assign adequate treatment aimed at prolonging the desired pregnancy.

Role of Toll Like Receptor 2 and 4 (TLR2 and TLR4) in Susceptibility and Resistance to Mycobacterium Tuberculosis Infection among Sudanese Patients

Background: Tuberculosis (TB), an infectious disease caused by Mycobacterium Tuberculosis (MTB). Toll-like receptors (TLRs) are the best-studied class of PRRs, and they recognize specific pathogen-associated molecular patterns (PAMPs) from various microorganisms. Objectives: This study aimed to determine and measure cellular TLR2 and TLR4 in active TB, latent TB and healthy individuals. Also aimed to define correlation between gene expression and present of TLR4 and TLR2 in study volunteers. Materials & Methods: This Multi-site, prospective unmatched age and sex case-control, hospital and community-based study. 244 Consented volunteersor patients diagnosed as active tuberculosis, diagnosed latent TBI and a community controls volunteers with negative both TST and INF γ release assay were enrolled. Three-color Flow cytometry using Heparinized blood to study surface expression of TLR4 (CD284) and TLR2 (CD282), by Mean channel fluorescence intensity (MFI). EDTA blood was used for mRNA expression of both receptors using Quantitative RT-PCR. Results: TLR2, TLR4 cell surface expression by Flow cytometry was decreased in all study volunteers with no significance differences (p.value 0.24).mRNA expression of TLR2 were increased in active TB compared to Latent TBI and healthy individuals,while mRNA TLR4 decreased in active TB and showed increased pattern in latent and low level of expression in healthy volunteers, associated with no significant difference in the expression of TLR-2 mRNA between patients and close contacts with LTBI (p. value 0.045), compared with significance correlation between cellular TLR2 and expression and BCG status (p. value 0.00) respectively. There was highly significance correlation between TST indurations and level of both TLR2 and TLR4 (p. value 0.00). Also there was significant correlation between mRNA expression of TLR2 and disease types (p value 0.01). Single Nucleotide Polymorphisms (SNPs) in TLR2and TLR4,in active Tb Patients Showed three alleles,group one were heterozygous allele, homozygous wild type in group two, while homozygous mutant present in group three with statistical significant with disease stages, (P.value 0.02), while LTBI volunteers had only homozygous mutant present and group two had undetermined alleles, and four of them develop active TB infection, while healthy controls had similar alleles. Conclusion: Up-regulation of TLR-2 and TLR-4 due to SNPs may be involved in the process of tuberculosis infection in Sudanese individuals.

Histone deacetylase inhibitors valproic acid and vorinostat enhance trastuzumab-mediated antibody-dependent cell-mediated phagocytosis

BackgroundThe monoclonal antibody (mAb) trastuzumab is part of the standard of care for patients with human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer. Antibody-dependent cell-mediated phagocytosis (ADCP) and cytotoxicity...

The Establishment of a Three-color Flow Cytometry Approach for the Scoring of Micronucleated Reticulocytes in Rat Bone Marrow

To develop and verify a flow cytometric measurement of reticulocytes (RETs) micronucleus in rat bone marrow. In our flow cytometric protocol, reticulocytes, leukocytes and DNA were labeled by anti-CD71-fluorescein isothiocyanate (FITC), anti-CD45-phycoerythrin (PE) and DRAQ5, respectively. Sprague-Dawley (SD) rats were assigned to four treatment groups randomly, and were exposed to ethyl methanesulfonate (EMS), cyclophosphamide (CP), ethyl nitrosourea (ENU) and colchicine (COL) respectively. Each treatment group was divided into four subgroups (5 rats per subgroup) according to different exposure dosage. A exposure dose of 0 was used as vehicle control for each group. Rats were administered with testing mutagens by gavage twice with a 24 h interval. Bone marrow from both femurs were collected 24 h after the last administration. The frequency of micronucleated reticulocytes (MN-RETs) and the percentage of reticulocytes (RETs%) were determined by flow cytometric measurement established in this study. And the manual counting method with microscope (by Giemsa staining) was conducted at the same time. A method for detection of reticulocyte micronucleus in bone marrow based on flow cytometry was successfully established. The MN-RETs in rat bone marrow of 20 SD rats treated by vehicle (i.e., background value of MN-RETs) was 0.83‰±0.12‰ by this method. The background value of MN-RETs in manual enumeration method was 1.43‰±0.44‰. It was obvious that the flow cytometric method had lower background value and more stable results. The trend, in which MN-RETs ascended and RETs% descended with increasing dose, can be detected by both methods in rats that exposed to EMS, CP, ENU and COL. Both methods were good to detect the correlation of induced-MN-RETs with four testing mutagens (the correlation coefficients were ranged from 0.834 3 to 0.913 7). With its sensitivity, rapidity, easy operation and low background value, the three-color flow cytometric enumerative protocol established in our laboratory can be used as a good substitute for manual micronucleus counting method and used in genotoxicity assessment of chemical substances.

Expression of CD44, CD87 and CD123 in Acute Leukemia and Its Correlation with Cellular Immunity

To investigate the expression of CD44, CD87 and CD123 in acute leukemia and its correlation with cellular immune markers. A total of 166 patients with acute leukemia (AL) admitted from May 2014 to February 2017 were enrolled in AL groups. Among these patients, 100 patients suffered from acute myeloid leukemia, 50 patients suffered from acute lymphoid leukemia, and 16 patients showed B/medullary phenotype. At the same time 50 patients with non-acute leukemia were enrolled in the control group. 5 ml of fasting venous blood collected from the patients in each group, and the percentage of CD44, CD87 and CD123 cells was determined by three-color flow cytometry. Symptomatic chemotherapy was given to the patients with confirmed acute leukemia, and the remission was evaluated after 2 treatmen courses. The Complete remission (CR) was recorded and the percentage of CD44, CD87 and CD123 cells under different curative efficacy were recorded. The correlation of the prognosis patients with CD44, CD87 and CD123 was analyzed by SPSS Pearson correlation analysis software. The positive rates of CD44, CD87 and CD123 in AL group were all higher than those in the control group (P＜0. 05). The positive rates of CD44 and CD123 in acute myeloid leukemia group were higher than those in acute lymphoblastic leukemia group and B/myeloid phenotype group (P＜0. 05). The positive rate of CD44 in acute lymphoid leukemia group was higher than that in B/medullary double phenotype group (P＜0.05). The treatment in the patients of AL group was successfully completed. 132 patients reachel to CR and 34 patients to PR+NR after 2 courses. The positive rates of CD44, CD87 and CD123 in CR patients were lower than those in PR+NR patients (P＜0.05). The results of SPSS Pearson correlation analysis showed that the prognosis of patients with acute leukemia negatively correlated with CD44 and CD87 (P＜0.05). The expression of CD44, CD87 and CD123 in different phenotype of acute leukemia are different, which correlateds with prognosis. The determination of CD44, CD87 and CD123 can be used to evaluate the prognosis of patients for the reference of clinical treatment.

Myeloid-Derived Suppressor Cells Promote Metastasis in Breast Cancer After the Stress of Operative Removal of the Primary Cancer.

Objective: To investigate the role of myeloid-derived suppressor cells (MDSC) in cancer progression after the stress of operative removal and the potential treatment value of MDSC depletion.Summary Background Data: Surgery is the most important treatment strategy in breast cancer. Recent research has provided evidence that operations may promote cancer metastases under some circumstances.Methods: A mouse model of breast cancer (administration of the murine breast cancer 4T1 cells subcutaneously) and the stress of operation were used to compare immune responses and survival outcomes. Flow cytometry was performed to detect the expression of CD11b and Gr1 MDSCs in tumor tissues and lung metastases. Cytokine levels were detected with three-color flow cytometry and enzyme-linked immunosorbent assay (ELISA). MDSCs were isolated and co-cultured with 4T1 cells to identify any morphological change with immunofluorescence. The anti Gr-1 antibody was used to detect the function of the anti-Gr1 treatment in breast cancer.Results: The operative stress impaired the overall survival, leading to an increased number of MDSCs that preferentially infiltrated the tumor microenvironment and promoted tumor metastasis. In both in vitro and in vivo assays, MDSCs induced the epithelial-mesenchymal transition (EMT) of tumor cells through the up-regulation of TGF-beta1, VEGF, and IL-10. Furthermore, a treatment strategy of MDSC depletion was found to reduce pulmonary metastases after operations.Conclusions: The stress of operation could impair the overall survival in mice. The infiltrated MDSCs appear to induce EMT of tumor cells and increase metastases through the up-regulation of TGF-beta1, VEGF, and IL-10 levels. MDSC depletion could be a promising treatment strategy to prevent immune evasion after operations.

Peripheral regulatory T cells and anti-inflammatory cytokines in children with juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIA) is a chronic, heterogenous inflammatory disease of unclear pathogenesis. JIA is hypothesized to be linked to a defective immune regulation. Anti-inflammatory cytokines belong to the best known regulatory factors. T-regulatory cells are a crucial cellular component of immune tolerance. One of their functions is synthesis of interleukin 10 (IL-10) and transforming growth factor beta1 (TGF-β1). The aim of this study was to determine the proportion of T-regulatory cells (CD4+CD25highFOXP3+) in peripheral blood, and serum levels of TGF-β1 and IL-10 in patients with JIA. The study included 25 patients with newly diagnosed JIA: oligoarthritis (n=17) and polyarthritis (n=8). The control group was comprised of 17 healthy children. CD4+CD25highFOXP3+ T cells in peripheral blood were quantified by means of three-color flow cytometry. Serum concentrations of TGF-β1 and IL-10 were estimated with ELISA. The proportion of peripheral CD4+CD25highFOXP3+ cells in patients with JIA was significantly higher than in the controls (p=0.04). The two groups did not differ significantly in terms of their TGF-β1 and IL-10 concentrations. At the time of diagnosis, children with JIA presented with an elevated proportion of T-regulatory cells (CD4+CD25highFOXP3+) in peripheral blood. Anti-inflammatory cytokines, IL-10 and TGF-β1, are not upregulated in the serum of patients with JIA, and therefore should not be considered as biomarkers of this condition.

Correlation between heat shock proteins, adiponectin, and T lymphocyte cytokine expression in type 2 diabetics.

Type 2 diabetes mellitus (T2DM) features insulin resistance, hyperglycemia, dyslipidemia, overproduction of inflammatory cytokines, and systemic oxidative stress. Here, heat shock proteins Hsp70 and Hsp 90, adiponectin, and heme oxygenase-1 (HO-1, Hsp32) are profiled in peripheral blood mononuclear cells (PBMC) and serum from 25 T2DM patients and 25 healthy control subjects. Cells cultured with phorbol 12-myristate 13-acetate/ionomycin were evaluated by three-color flow cytometry for immunophenotypic biomarkers. Plasma HO-1, Hsp, and adiponectin levels were assayed by enzyme-linked immunosorbent assay (ELISA). Relative to healthy controls, T2DM patients exhibited significantly elevated plasma Hsp70, and representation of T helper immunophenotypes activated to express inflammatory cytokines, including CD4+ IFN-γ+, CD4+ TNF-α+, CD4+ IL-6+, CD4+ IL-1β+ T cells, significantly lower representation of CD4+ IL-10+ T cells, plasma adiponectin and cell-associated HO-1 expression-with no significant differences in plasma Hsp90 between T2DM and healthy controls. Plasma HO-1 and adiponectin in T2DM patients inversely correlated with TNF-α and showed inverse correlation between serum LDL and plasma HO-1. Moreover, TNF-α and Hsp90 in T2DM patients correlated positively with fasting blood glucose (FBG). These results demonstrate correlation between potentially pathogenic T cells, HO-1, and adiponectin, additionally revealing a T helper (Th)1-related character of T2DM immunopathogenesis, suggesting potential for novel T cell-related management strategies for T2DM and related co-morbidities.

Analysis of PD-1 and Tim-3 expression on CD4+ T cells of patients with rheumatoid arthritis; negative association with DAS28.

Expression of T cell immunoglobulin and mucin-domain containing-3 (Tim-3) and programmed cell death-1 (PD-1) was studied on CD4+ T cells of patients with rheumatoid arthritis (RA). Association of Tim-3 and PD-1 expression with disease activity of RA patients was also addressed. A total of 37 RA patients and 31 sex- and age-matched healthy controls were included in this study. Disease activity of RA patients was determined by Disease Activity Score of 28 joints scoring system (DAS28). A three-color flow cytometry method was applied to determine the frequency of Tim-3+/PD-1+/CD4+ T cells. To measure the cytokine production, peripheral blood mononuclear cells (PBMCs) were stimulated with PMA/ionomycin. Concentrations of IL-17, IL-10, IFN-γ, and TNF-α were measured in culture supernatants by ELISA. The frequency of PD-1+/CD4+ and Tim-3+/PD-1+/CD4+ T cells was significantly higher in patients with RA compared to that in controls (p = 0.0013 and p = 0.050, respectively). The percentage of Tim-3+/CD4+ T cells was similar in patients and controls (p = 0.4498). The RA patients have produced significant higher levels of TNF-α, IL-17, and IFN-γ than those of healthy controls (p = 0.0121, p = 0.0417, and p = 0.0478, respectively). Interestingly, an inverse correlation was found between the frequency of Tim-3+/CD4+ cells and DAS28 of RA patients (r = - 0.4696, p = 0.0493). Similarly, the percentage of Tim-3+/PD-1+/CD4+ T cells was also revealed an inverse correlation with DAS28 (r = - 0.5268, p = 0.0493). Moreover, significant positive correlations were detected between the concentrations of TNF-α (r = 0.6418, p = 0.0023) and IL-17 (r = 0.4683, p = 0.0373) with disease activity of RA patients. Our results indicate that Tim-3 and PD-1 are involved in immune dysregulation mechanisms of rheumatoid arthritis and could be considered as useful biomarkers for determination of disease activity and progression.

Desert hedgehog knockout mice show a reduced ratio of CD4+:CD8+ in thymic developing T cells

&lt;p class="abstract"&gt;&lt;strong&gt;Background:&lt;/strong&gt; Thymic differentiation is important and determines the strength of adaptive immunity in mammals in their later days in life. There are many factors that have been found to influence the development of T lymphocytes in the thymus and these include the effect of hedgehog signalling family of proteins. Immunologists and other basic science researchers have established the role of Indian hedgehog and sonic hedgehog in thymocytes development in the recent past, but the role of hedgehog is not well known. The aim of this study was to determine the influence of Desert hedgehog in CD4:CD8 ratio in developing thymocytes of mice.&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Methods:&lt;/strong&gt; Smashed thymocytes from mice deficient of Desert hedgehog and those with an intact gene coding for this protein were prepared in a cell suspension using standard procedurs. The cell suspensions were stained using fluorochrome-labelled monoclonal anti: CD4-PE, CD8-TRI, CD3-FITC, CD5-FITC, CD44-PE and CD25-FITC (e-Bioscience). Samples were analyzed using a three-color flow cytometry. The flow cytometry-generated data was analyzed using flowjo (Treestar, USA). Statistical significance of the findings was determined using paired t-test and reported at p&amp;lt;0.05. &lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Results:&lt;/strong&gt; There was a general upward trend on CD4+CD8+ double positive thymocytes in Desert hedgehog mice relative to WT controls. An analysis of CD4:8 ratio revealed a reduced ratio in Dhh KO mice compared to that of WT controls attributable to the finding that there might have been a preferential differentiation of DP CD4+CD8+ to SP CD4+ in Dhh knockout mice as demonstrated by percentage of thymic subsets. The results of this study were not statistically significant and this was blamed on the fewer number of animals used in the study.&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Conclusions:&lt;/strong&gt; Dhh might have a role arresting the DP cell subjects from differentiating preferentially to CD4+ T cells. To get statistically significant findings, these experiments could be repeated with a larger animal sample.&lt;/p&gt;

Peripheral and Intestinal T-regulatory Cells are Upregulated in Children with Inflammatory Bowel Disease at Onset of Disease

ABSTRACTBackground/Aims: To determine the proportion of T-regulatory cells (CD4+CD25highFOXP3+ cells) in peripheral blood and the number of FOXP3+ cells in intestinal mucosa of children with inflammatory bowel disease (IBD), and to verify whether these parameters correlate with the activity of the disease.Material and methods: 24 patients newly diagnosed for IBD were included in the study: ulcerative colitis (UC; n = 13) and Crohn’s disease (CD; n = 11). Seventeen healthy controls (HC) and 16 patients with irritable bowel syndrome (IBS) served as a control group for peripheral and intestinal Tregs assessment, respectively. The disease activity was assessed by Pediatric Ulcerative Colitis Activity Index (PUCAI) and Pediatric Crohn’s Disease Activity Index (PCDAI). Quantification of regulatory T cells of CD4+CD25highFOXP3+ phenotype in peripheral blood was based on three-color flow cytometry. Mucosal Tregs represented by FOXP3+ cells were evaluated using immunohistochemistry.Results: Median proportion of CD4+CD25highFOXP3+ cells among CD4+ T cells in peripheral blood (5.1%, range 1.7–84% vs. 4.3%, range 2–8.1%, p = 0.023) and median number of intestinal FOXP3+ cells (115.33 per high-power field, hpf, range 39.33–375.67 vs. 10.16 per hpf, range 5–30, p = 0.0001) were significantly higher in children with IBD than in the controls. The proportion of circulating Tregs and the number of intestinal FOXP3+ cells did not correlate with clinical activity of the disease, as well as with endoscopic and histopathologic scoring. No significant correlation was found between the percentage of peripheral CD4+CD25highFOXP3+ cells and the number of intestinal FOXP3+cells.Conclusions: Children with IBD likely do not present with a quantitative deficiency of circulating and intestinal Tregs at the moment of diagnosis.

PP.04.05] ACCELERATED DEVELOPMENT OF IMMUNE CELLS IN CHILDREN WITH PRIMARY HYPERTENSION

Objective: Immune system plays a role in pathogenesis of primary hypertension (PH) and activation of immune cells is associated with subclinical hypertensive arterial injury. One of the markers of maturation of immune cells is increased number and percentage of memory cells. We hypothesized that children with PH have faster maturation of immune cells. Design and method: The distribution of peripheral T cell subsets (CD4+ and CD8+) expressing CD45 common leukocyte antigen (CD45Ag) isoforms: CD45RA (naive cells), CD45RO (memory cells) and CD31 (pecam-1, a marker of early thymic emigrants) was evaluated by three-color flow cytometry in a group of 11 PH adolescents and 16 healthy, age matched controls. Results: Hypertensive children had higher proportion of “memory” CD8 cells (CD8/CD8RO+) than their healthy counterparts (17.0 range: 11.9–23.8 vs 10.9 9.1–14.1; p = 0.03), lower of CD8/CD8RA+ cells (72.3 range: 66.5–81.9 vs 81.6 77.4–84.3; p = 0.05), and lower CD8RA/CD8RO+ ratio than the controls (5.1 range: 2.7–6.9 vs 7.4 5.5–8.9; p = 0.028). Percentage of CD4 and CD8 T bearing “naive” or “memory” phenotype correlated with brachial and aortic pulse pressure. Additionally, CD8RA/RO+ ratio was associated with pulse pressure (p = 0.03, r = −0.337) and aortic pulse pressure (p = 0.03, r = −0.384). There was also negative correlation of percentage of CD4/CD31+ with both absolute and standardized values of carotid wall cross sectional area (p = 0.01, r = −0.433 p = 0.016, r = −0.410, respectively). Conclusions: Adolescents with PH present subtle alterations in maturation of immune system manifested by: a) the decreased number of early thymic emigrants within CD4+ T cell suggesting accelerated thymic senescence, b) the increased population of more mature cells expressing “memory” markers, c) the associations with indirect indices of arterial stiffness and carotid artery remodelling and d) shift towards “memory” phenotype in T cells. Altogether these findings indicate that early stage of human primary hypertension and arterial remodelling may be related to certain defects in T cell development that result in accelerated maturation and senescence of immune system.

Regulatory T Cells in Patients with Idiopathic Thrombocytopenic Purpura.

Objective:Immune thrombocytopenic purpura (ITP) is an immune-mediated bleeding disorder in which platelets are opsonized by autoantibodies and destroyed by an Fc receptor-mediated phagocytosis by the reticuloendothelial system within the spleen. Autoimmune processes are also considered in the pathogenesis of this disorder. CD4+CD25+FoxP3+ regulatory T (Treg) cells and CD8+CD28- Treg cells have roles in autoimmune diseases. We investigated these regulatory cells in ITP patients.Materials and Methods:We included 22 ITP patients and 16 age-matched healthy subjects. CD4+CD25+FoxP3+ Treg cells and CD8+CD28- cells were investigated by three-color flow cytometry. The ratios of these cell populations to total lymphocytes were calculated. Statistical analysis was carried out with the Mann-Whitney U test.Results:CD4+CD25+ Treg cells were 9.69±3.70% and 12.99±5.58% in patients with ITP and controls, respectively. CD4+CD25highFoxP3+ cells were 27.72±19.74% and 27.55±23.98% in ITP patients and controls, respectively. The percentages of both of these cell types were not statistically significant when compared to the control group.Conclusion:We did not find any differences in ratios of CD4+CD25+FoxP3+ Treg cells or CD8+CD28- T cells in lymphocytes between patients and healthy subjects. We conclude that these circulatory cells are not different in ITP, but further studies are needed to explore the putative roles of these regulatory cells.

Toll-like receptors 7 and 8 mRNA in monocytes of Egyptian children with chronic hepatitis C infection genotype 4 as predictor to “response” interferon and ribavirin treatment

BackgroundThe contribution of the host immune response to sustained virologic response (SVR) is not clear in children with chronic hepatitis C (CHC). It is still unknown whether TLRs 7&8 expression in monocytes is related to disease progression in HCV infected children or whether it represents a protective factor in those patients who are slow disease-progressors. ObjectiveThe aim of this study was to assess the value of expression of TLRs 7&8 and TNF as a predictor for the outcome of antiviral therapy in children with CHC. Patients and methodsThis prospective study included fifty CHC children aged 5–18years. Thirty-four were males, sixteen were females who received Peg-IFN-α-2b and RBV for 48weeks. mRNA levels of TLRs 7&8 in monocytes were evaluated using real-time PCR before a 48-week treatment with Peg-IFN-α-2b and RBV. In addition, we studied tumor necrosis factor (TNF) production in monocytes using three-color immunofluorescence and flow cytometry after TLRs 7&8 ligand Resiquimod (R848) stimulation. ResultsAccording to the virological response of the treatment, the infected children were classified into sustained virological response (SVR) 72% and non-responder 28%. The study determined that mRNA levels of TLRs 7&8 were significantly high in SVR compared to non-responders. Furthermore, after stimulation with R848 the median fluorescence intensity (MFI) for TNF protein was significantly high in SVR compared to non-responders. ConclusionsTLRs 7&8 and TNF have great importance in controlling HCV infection and the responses to IFN therapy. Further research covering other stages of CHC at large numbers and at different monocytes subsets levels will be needed.

Clinical Significance of Immunophenotyping of Bone Marrow Cells in Multiple Myeloma

Aim. To analyze the relationship between expression of aberrant CD45, CD19, CD56 markers on the plasma cells and clinical and laboratory findings and prognostically significant parameters in patients with multiple myeloma (MM). Methods. This scientific research includes data on clinical investigation and immunophenotyping of bone marrow cells obtained from 64 MM patients treated in the N.N. Blokhin Russian Cancer Research Center over the period from 2004 to 2015. The three-color flow cytometry was performed using a direct immunofluorescence technique (CD38-PerCP, CD138-FITC monoclonal antibodies) and PE-conjugated monoclonal antibodies against CD45, CD19, and CD56. Results. Comparison of average values of the total count of plasma cells, the number of plasmablasts, proplasmacyte and mature plasma cells (according to the myelogram) and comparison of these data with the level of expression of the CD19 marker demonstrated a significant relationship between the CD19 negative immunophenotype and both a higher level of the total count of plasma cells and immature plasma cells. There also was a significant correlation between the CD19 negative immunophenotype and a higher level of C-reactive protein, which is significant prognostic factor in MM. In addition, there was a significant relationship between the CD19 negative phenotype and a higher percentage of young neutrophils in blood, i.e. with a more frequent “left shift”. The CD56 negative phenotype is associated with plasmablastic morphology of plasma cells and with the presence of plasma cells in the peripheral blood. Plasma cell leukemia is more common in patients with CD56 negative phenotype of myeloma cells. The CD45 negative immunophenotype was associated with a higher level of к-type FLCs, Bence-Jones proteinuria and with a higher serum creatinine, than in the cases of CD45 positive phenotype. Conclusion. The study of the immunophenotype of plasma cells in MM has important scientific and practical significance and requires further study.

Alterations in immunophenotype of peripheral blood mononuclear cells in children with chronic viral hepatitis following anti-viral therapy

The aim of this study was to investigate the impact interferon type I based anti-viral therapy on the immune system of chronically infected children with hepatitis C and B virus. Peripheral blood mononuclear cells were examined by three-color flow cytometry, using a panel of fluorochrome labeled monoclonal antibodies. It was found that the majority of white blood cell subsets were decreased in patients after termination of long-term anti-viral treatment. In particular, potentially cytotoxic, effector cell subsets, such as natural killer, natural killer T-lymphocytes and CD8+CD28- T cells were significantly decreased. Moreover, plasmacytoid dendritic cells were decreased, both in percentage and in absolute values. These data suggest that interferon-α based treatment, apart from its anti-viral effect, has also remarkable impact on patient's cell-mediated immunity.