Foxp3+CD39+CD73+ regulatory T-cells are decreased in the peripheral blood of women with deep infiltrating endometriosis.

Endometriosis's pathophysiology remains incompletely understood, with evidence pointing towards a dysregulated immune response. Regulatory T (Treg) cells, pivotal in maintaining self-tolerance, may facilitate the survival of ectopic endometrial cells within the abdominal cavity, thereby contributing to endometriosis development. This study aimed to assess the prevalence of CD39+CD73+ suppressor Treg cell subsets in the peripheral blood of endometriosis patients. This research focuses on the pivotal role of regulatory T-cells (Tregs), which are essential for maintaining immune tolerance and preventing autoimmune diseases. A case-control study was conducted, including 32 women diagnosed with endometriosis and 22 control subjects. The frequency of peripheral blood CD39+CD73+ suppressor Treg cells was quantified using flow cytometry. No significant differences were observed in the frequency of CD3+CD4+CD25High cells (Median [M]: 10.1; Interquartile Range [IQR]: 6.32‒18.3 vs. M: 9.72; IQR: 6.22-19.8) or CD3+CD4+CD25HighCD39+Foxp3+ cells (M: 31.1; IQR: 19.7-44.0 vs. M: 30.55; IQR: 18.5-45.5) between controls and patients. However, a significantly lower frequency of CD3+CD4+CD25HighCD39+CD73+ cells was observed in the endometriosis group compared to controls (M: 1.98; IQR: 0.0377-3.17 vs. M: 2.25; IQR: 0.50-4.08; p = 0.0483), suggesting a reduction in systemic immune tolerance among these patients. This finding highlights the potential role of CD39 and CD73 expression on Treg cells as biomarkers for assessing disease severity and progression. Furthermore, elucidating the mechanisms driving these alterations may unveil new therapeutic strategies to restore immune equilibrium and mitigate endometriosis symptoms.

Genetics and regulation of HDL metabolism.

The inverse association between plasma HDL cholesterol (HDL-C) levels and risk for cardiovascular disease (CVD) has been demonstrated by numerous epidemiological studies. However, efforts to reduce CVD risk by pharmaceutically manipulating HDL-C levels failed and refused the HDL hypothesis. HDL-C levels in the general population are highly heterogeneous and are determined by a combination of genetic and environmental factors. Insights into the causes of HDL-C heterogeneity came from the study of monogenic HDL deficiency syndromes but also from genome wide association and Μendelian randomization studies which revealed the contribution of a large number of loci to low or high HDL-C cases in the general or in restricted ethnic populations. Furthermore, HDL-C levels in the plasma are under the control of transcription factor families acting primarily in the liver including members of the hormone nuclear receptors (PPARs, LXRs, HNF-4) and forkhead box proteins (FOXO1-4) and activating transcription factors (ATFs). The effects of certain lipid lowering drugs used today are based on the modulation of the activity of specific members of these transcription factors. During the past decade, the roles of small or long non-coding RNAs acting post-transcriptionally on the expression of HDL genes have emerged and provided novel insights into HDL regulation and new opportunities for therapeutic interventions. In the present review we summarize recent progress made in the genetics and the regulation (transcriptional and post-transcriptional) of HDL metabolism.

Screening of 23 candidate genes by next-generation sequencing of patients with permanent congenital hypothyroidism: novel variants in TG, TSHR, DUOX2, FOXE1, and SLC26A7.

PURPOSE: To date, many genes have been associated with congenital hypothyroidism (CH). Our aim was to identify the mutational spectrum of 23 causative genes in Turkish patients with permanent CH, including thyroid dysgenesis (TD) and dyshormonogenesis (TDH) cases. METHODS: A total of 134 patients with permanent CH (130 primary, 4 central) were included. To identify the genetic etiology, we screened 23 candidate genes associated with CH by next-generation sequencing. For confirmation and to detect the status of the specific familial variant in relatives, Sanger sequencing was also performed. RESULTS: Possible pathogenic variants were found in 5.2% of patients with TD and in 64.0% of the patients with normal-sized thyroid or goiter. In all patients, variants were most frequently found in TSHR, followed by TPO and TG. The same homozygous TSHB variant (c.162 + 5G > A) was identified in four patients with central CH. In addition, we detected novel variants in the TSHR, TG, SLC26A7, FOXE1, and DUOX2. CONCLUSION: Genetic causes were determined in the majority of CH patients with TDH, however, despite advances in genetics, we were unable to identify the genetic etiology of most CH patients with TD, suggesting the effect of unknown genes or environmental factors. The previous studies and our findings suggest that TSHR and TPO mutations is the main genetic defect of CH in the Turkish population.

Successful Milk Oral Immunotherapy Promotes Generation of Casein-Specific CD137+ FOXP3+ Regulatory T Cells Detectable in Peripheral Blood.

Background: Oral immunotherapy (OIT) is an emerging treatment for cow's milk protein (CMP) allergy in children. The mechanisms driving tolerance following OIT are not well understood. Regulatory T cells (TREG) cells are key inhibitors of allergic responses and promoters of allergen-specific tolerance. In an exploratory study, we sought to detect induction of allergen-specific TREG in a cohort of subjects undergoing OIT. Methods: Pediatric patients with a history of allergic reaction to cow's milk and a positive Skin Pick Test (SPT) and/or CMP-specific IgE >0.35 kU, as well as a positive oral challenge to CMP underwent OIT with escalating doses of milk and were followed for up to 6 months. At specific milestones during the dose escalation and maintenance phases, casein-specific CD4+ T cells were expanded from patient blood by culturing unfractionated PBMCs with casein in vitro. The CD4+ T cell phenotypes were quantified by flow cytometry. Results: Our culture system induced activated casein-specific FOXP3+Helios+ TREG cells and FOXP3- TEFF cells, discriminated by expression of CD137 (4-1BB) and CD154 (CD40L) respectively. The frequency of casein-specific TREG cells increased significantly with escalating doses of milk during OIT while casein-specific TEFF cell frequencies remained constant. Moreover, expanded casein-specific TREG cells expressed higher levels of FOXP3 compared to polyclonal TREG cells, suggesting a more robust TREG phenotype. The induction of casein-specific TREG cells increased with successful CMP desensitization and correlated with increased frequencies of casein-specific Th1 cells among OIT subjects. The level of casein-specific TREG cells negatively correlated with the time required to reach the maintenance phase of desensitization. Conclusions: Overall, effective CMP-OIT successfully promoted the expansion of casein-specific, functionally-stable FOXP3+ TREG cells while mitigating Th2 responses in children receiving OIT. Our exploratory study proposes that an in vitro TREG response to casein may correlate with the time to reach maintenance in CMP-OIT.

Infants Younger Than 90 Days Admitted for Late-Onset Sepsis Display a Reduced Abundance of Regulatory T Cells.

Objective: To provide epidemiological data of infants < 90 days of age with suspected late-onset sepsis (LOS) and evaluate distinct immunological specificities. We hypothesized that previously healthy infants < 3 months of age with sepsis have a yet undefined immunological predisposition; e.g. differences in lymphocyte subsets including regulatory T cells. Methods: We performed an exploratory, single center study between January 1st, 2019 and June 1st, 2021. Routine diagnostics included conventional culture (blood, cerebrospinal fluid, urine), PCR and inflammatory markers in infants < 90 days of age with suspected sepsis. We additionally analyzed lymphocyte subsets and CD4+ CD25+ forkhead box protein (FoxP3)+ Tregs at admission for sepsis workup as compared to age-matched controls. Results: A convenience sample cohort of n= 51 infants with sepsis workup was enrolled. Invasive bacterial infection (IBI) was diagnosed in 25 (49.0%) patients including two infants with a rhinovirus co-infection and viral infection in 14 (27.5%) neonates. No infectious cause was found in 12 cases. Infants with suspected LOS displayed a decreased abundance of CD4+ FoxP3+ T cells as compared to controls, which was most pronounced in the subgroup of infants with IBI. We also noticed elevated HLA-DR-positive CD3+ cells in infants with LOS and a higher CD4/CD8-ratio in infants with viral infection as compared to healthy controls. Infants with viral infections had a higher number of natural killer cells as compared to infants with IBI. Conclusion: Our exploratory data support the concept of a potential immaturity state and failed immune tolerance development for young infants with LOS. Future large-scale studies are needed to elucidate pre-sepsis conditions and to target the microbiome-immunity interplay as a potential risk pattern.

Circulating CD4+ FoxP3- CXCR5- CXCR3+ PD-1hi cells are elevated in active rheumatoid arthritis and reflect the severity of the disease.

OBJECTIVE: To examine the expression and clinical significance of circulating CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in rheumatoid arthritis (RA). METHODS: CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in peripheral blood of 35 patients with active RA, 17 with RA in stable remission, and 24 healthy controls were analyzed by flow cytometry. Serum IgG and circulating plasmablast percentages were measured and correlations with CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells were systematically analyzed. Disease Activity Scale 28 (DAS28) scores were also calculated and correlation analysis with CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells was conducted. The levels of CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells were compared before and after disease-modifying anti-rheumatic drug treatment. Cytokine levels in plasma and cytokine secretion in CD4 cells were measured and their correlations with CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells were further analyzed. RESULTS: The levels of CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in the peripheral blood of patients with active RA were significantly increased compared with healthy controls. CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in patients with active RA were positively correlated with serum IgG and DAS28 scores. CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells were significantly decreased in patients after treatment. Plasma interleukin-10 concentrations and interleukin-10-positive CD4 cell percentages were significantly positively correlated with CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cell levels. CONCLUSION: Circulating CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in patients with active RA are increased and could reflect the severity of the disease, which may play a potential role in the pathogenesis of RA.

Genome wide DNA methylation analysis of alveolar capillary dysplasia lung tissue reveals aberrant methylation of genes involved in development including the FOXF1 locus.

BACKGROUND: Alveolar capillary dysplasia with or without misalignment of the pulmonary veins (ACD/MPV) is a lethal congenital lung disorder associated with a variety of heterozygous genomic alterations in the FOXF1 gene or its 60 kb enhancer. Cases without a genomic alteration in the FOXF1 locus have been described as well. The mechanisms responsible for FOXF1 haploinsufficiency and the cause of ACD/MPV in patients without a genomic FOXF1 variant are poorly understood, complicating the search for potential therapeutic targets for ACD/MPV. To investigate the contribution of aberrant DNA methylation, genome wide methylation patterns of ACD/MPV lung tissues were compared with methylation patterns of control lung tissues using the recently developed technique Methylated DNA sequencing (MeD-seq). RESULTS: Eight ACD/MPV lung tissue samples and three control samples were sequenced and their mutual comparison resulted in identification of 319 differentially methylated regions (DMRs) genome wide, involving 115 protein coding genes. The potentially upregulated genes were significantly enriched in developmental signalling pathways, whereas potentially downregulated genes were mainly enriched in O-linked glycosylation. In patients with a large maternal deletion encompassing the 60 kb FOXF1 enhancer, DNA methylation patterns in this FOXF1 enhancer were not significantly different compared to controls. However, two hypermethylated regions were detected in the 60 kb FOXF1 enhancer of patients harbouring a FOXF1 point mutation. Lastly, a large hypermethylated region overlapping the first FOXF1 exon was found in one of the ACD/MPV patients without a known pathogenic FOXF1 variation. CONCLUSION: This is the first study providing genome wide methylation data on lung tissue of ACD/MPV patients. DNA methylation analyses in the FOXF1 locus excludes maternal imprinting of the 60 kb FOXF1 enhancer. Hypermethylation at the 60 kb FOXF1 enhancer might contribute to FOXF1 haploinsufficiency caused by heterozygous mutations in the FOXF1 coding region. Interestingly, DNA methylation analyses of patients without a genomic FOXF1 variant suggest that abnormal hypermethylation of exon 1 might play a role in some ACD/MPV in patients.

Identification of circulating regulatory T lymphocytes with membrane markers - a new multiparameter flow cytometry protocol.

INTRODUCTION: Regulatory T cells (Tregs) are a unique CD4+ T cell subset involved in the regulation of immune responses. The traditional immunophenotype used to define Tregs includes CD4+CD25high and the expression of the transcription factor Forkhead box protein 3 (FoxP3). A complex technique of intracellular staining, transient upregulation of FoxP3 in activated conventional T lymphocytes (Tcons), and the omission of naïve CD45RA+ Tregs with downregulated FoxP3 activity but a demethylated FOXP3 promoter region may lead to inaccurate quantification. In an attempt to meet the need for a reliable and simplified enumeration strategy, we investigated different membrane markers to capture the entire Treg compartment and to identify subpopulations of Tregs. MATERIAL AND METHODS: Analyses were performed on whole blood. Tested gating strategies were based on the expression of the following membrane antigens: CD45, CD3, CD4, CD25, CD127, CD26, CD6, CD39, CD71, HLA-DR, CD45RA and CD31. Double controls with FoxP3 were performed. RESULTS: The final enumeration panel consisted of the membrane markers CD45, CD3, CD4, CD25, CD127, CD26, CD39, CD45RA and CD31. A deep analysis of T cells with the CD4+CD25+CD127low/-CD26low/-CD45RAimmunophenotype revealed high expression of FoxP3 and/or CD39, while cells with the naïve immunophenotype, CD4+CD25+CD127low/-CD26low/-CD45RA+, presented lower expression of suppressor markers. Antigen CD31 is considered to be a valuable membrane marker of thymus-derived Tregs. CONCLUSIONS: The presented 9-color panel that can be easily applied in laboratories enables reliable enumeration of Tregs with additional information about the functionality, maturity and origin of T regulatory cells.

Differences in Maturation Status and Immune Phenotypes of Circulating Helios+ and Helios- Tregs and Their Disrupted Correlations With Monocyte Subsets in Autoantibody-Positive T1D Individuals.

CD4 Tregs are involved in the regulation of various autoimmune diseases but believed to be highly heterogeneous. Studies have indicated that Helios controls a distinct subset of functional Tregs. However, the immunological changes in circulating Helios+ and Helios- Tregs are not fully explored in type 1 diabetes (T1D). Here, we elucidated the differences in maturation status and immune regulatory phenotypes of Helios+ and Helios- Tregs and their correlations with monocyte subsets in T1D individuals. As CD25-/low FOXP3+ Tregs also represent a subset of functional Tregs, we defined Tregs as FOXP3+CD127-/low and examined circulating Helios+ and Helios- Treg subpopulations in 68 autoantibody-positive T1D individuals and 68 age-matched healthy controls. We found that expression of both FOXP3 and CTLA4 diminished in Helios- Tregs, while the proportion of CD25-/low Tregs increased in Helios+ Tregs of T1D individuals. Although the frequencies of neither Helios+ nor Helios- Tregs were affected by investigated T1D genetic risk loci, Helios+ Tregs correlated with age at T1D diagnosis negatively and disease duration positively. Moreover, the negative correlation between central and effector memory proportions of Helios+ Tregs in healthy controls was disrupted in T1D individuals. Finally, regulatory non-classical and intermediate monocytes also decreased in T1D individuals, and positive correlations between these regulatory monocytes and Helios+/Helios- Treg subsets in healthy controls disappeared in T1D individuals. In conclusion, we demonstrated the alternations in maturation status and immune phenotypes in Helios+ and Helios- Treg subsets and revealed the missing association between these Treg subsets and monocyte subsets in T1D individuals, which might point out another option for elucidating T1D mechanisms.

Effect of intraoperative blood transfusion on Treg and FOXP3 in patients with digestive tract malignancies and different ABO blood types.

BACKGROUND: Blood transfusion can cause immunosuppression and lead to worse outcomes in patients with digestive tract malignancies; however, the specific mechanism behind this is not completely understood. One theory is that increased numbers of regulatory CD3+CD4+CD25+FOXP3+ T cells (Tregs) and forkhead box protein-3 mRNA (FOXP3) expression in the blood after transfusion contribute to these outcomes. The effect of blood transfusion on immune function in patients with different ABO blood types is variable. This study investigates the effect of intraoperative blood transfusion on the number of Tregs and the expression of FOXP3 in the blood of patients with different ABO blood types and digestive tract malignancies. METHODS: Patients with digestive tract malignancies who underwent radical resection and received intraoperative blood transfusion were divided into four groups according to their blood types:blood group A, blood group B, blood group O and blood group AB (n = 20 for each group). Blood was collected from all patients before surgery, immediately after transfusion, 1 day after transfusion, and 5 days after transfusion. The number of Tregs was measured by flow cytometry. The expression of FOXP3 was detected by real time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: There was no significant difference in the number of Tregs or expression of FOXP3 mRNA among patients with different blood types before surgery. However, the number of Tregs and the expression of FOXP3 increased after blood transfusion in all blood type groups. This increase was especially evident and statistically significant on the first day after blood transfusion when compared with measures obtained before the surgery. Measures returned to the preoperative level five days after surgery. There were significant differences in the increase of Tregs and expression of FOXP3 among patients with different blood types. The greatest increase was seen in patients with blood group B and the least in blood group A. CONCLUSIONS: Intraoperative blood transfusion can lead to an increase in blood Tregs and FOXP3 expression in patients with digestive tract malignancies. Increases were greatest on the first day after surgery and differed among patients with different blood types. Increases were greatest in blood type B and least in blood type A.

Correlations of Helicobacter pylori with liver function, inflammatory factors and serum levels of FoxP3 and RORγt in patients with hepatitis B cirrhosis.

OBJECTIVE: To investigate the correlations of Helicobacter pylori (HP) with liver function, inflammatory factors and serum levels of forkhead box P3 (FoxP3) and retinoic acid receptor-related orphan receptor gamma-t (RORγt) in patients with hepatitis B cirrhosis (HBC). PATIENTS AND METHODS: A total of 60 HBC patients were divided into HBC group (n=30) and HP-infected HBC group (HP&HBC group, n=30). QRT-PCR was conducted to determine the messenger ribonucleic acid (mRNA) levels of FoxP3 and RORγt in serum samples. ELISA was applied to measure the levels of relevant inflammatory factors. Besides, immunohistochemical staining was conducted to detect positive expressions of FoxP3 and RORγt in liver tissues of patients in the two groups. RESULTS: No significant differences in gender, drinking, smoking, diabetes and age were found between HBC group and HP&HBC group (p>0.05). Globulin and albumin levels were comparable between the two groups (p>0.05). Liver function indexes, including ALT, AST and TBIL were higher in HP&HBC group than those in HBC group (p<0.05). The HBV-DNA level was lower in HBC group in comparison with that in HP&HBC group. The interferon-gamma (IFN-γ) level was remarkably higher in HBC group than that in HP&HBC group (p<0.01), and the levels of interleukin (IL)-6, IL-10, IL-17 and transforming growth factor (TGF)-ß1 were notably lower in HBC group in comparison with those in HP&HBC group (p<0.01). Additionally, the mRNA levels of FoxP3 and RORγt in HBC group were distinctly lower than those in HP&HBC group (p<0.01). The mRNA levels of FoxP3 and RORγt were positively related to those of IL-6, IL-10, IL-17, and TGF-ß1, and negatively associated with IFN-γ level. Immunohistochemical results indicated that positive expression rates of FoxP3 and RORγt in the liver tissues were approximately 50% in HP&HBC group and B. Zhao, Q.-J. Sheng, Y. Qin, X.-L. Wang, H. Zhao, N. Zhaowere 15% in HBC group, and the difference was statistically significant (p<0.05). CONCLUSIONS: Expression levels of FoxP3 and RORγt in serum and liver tissues are elevated in HP-infected HBC patients, and inflammatory factors are correlated with their expressions, suggesting the aggravated liver damage.

Foxp3 TSDR Hypermethylation Is Correlated with Decreased Tregs in Patients with Unexplained Recurrent Spontaneous Abortion.

A decline of T regulatory cell (Treg) number and function is associated with unexplained recurrent spontaneous abortion (URSA). However, the mechanism of downregulation of Tregs in URSA patients is still unknown. This study aimed to investigate the changes of Tregs in URSA patients and the epigenetic regulation for these changes. Venous blood samples were collected from 20 patients with URSA and 20 healthy control subjects. Treg number and inhibitory capacity, and Foxp3 mRNA expression and Foxp3 TSDR methylation were compared between the 2 groups. Correlations between Treg frequency and inhibitory function and TSDR methylation status were examined by Spearman's correlation. The proportion of Tregs within the population of CD4+ T cells and the expression of Foxp3 mRNA was significantly lower in URSA patients than in healthy control subjects. Tregs from URSA patients and healthy controls both significantly inhibited the cytotoxic activity of natural killer (NK) cells toward K562 targets; however, the inhibitory ability of Tregs from URSA patients was significantly lower than that from healthy controls. The methylation level of the Treg-specific demethylated region (TSDR) in the Foxp3 gene was significantly greater in URSA patients than in the controls, and the level of methylation was inversely correlated with the proportion of Tregs and Foxp3 mRNA expression in the peripheral blood. However, the methylation level was not correlated with the inhibitory function of Tregs. A decrease of Treg number and function may be related to the pathogenesis of URSA, and Foxp3 hypermethylation may be associated with the decreased Treg number.

miR-375-3p contributes to hypoxia-induced apoptosis by targeting forkhead box P1 (FOXP1) and Bcl2 like protein 2 (Bcl2l2) in rat cardiomyocyte h9c2 cells.

miRNAs have been pointed to play critical role in the development of congenital heart disease (CHD). miRNA-375-3p (miR-375-3p) was involved in cardiac dysfunction and cardiogenesis. However, no prior study had established a therapeutic role of miR-375-3p in CHD. We intended to investigate the effect and mechanism of miR-375-3p on apoptosis in hypoxic cardiomyocytes in vitro. Expression of miR-375-3p, forkhead box P1 (FOXP1) and Bcl2 like protein 2 (Bcl2l2) was detected using real-time quantitative PCR and western blot. Apoptosis was measured with MTT assay, flow cytometry and caspase-3 activity assay. The potential target binding between miR-375-3p and FOXP1/Bcl2l2 was predicted on DianaTools, and was validated by luciferase reporter assay and RNA pull-down assay. As a result, miR-375-3p was upregulated and FOXP1/Bcl2l2 was downregulated in maternal serum of women with fetal CHD and hypoxia-induced rat cardiomyocyte h9c2 cells. Hypoxia induced apoptosis rate elevation, caspase-3 activity promotion and viability inhibition in h9c2 cells; overexpression of miR-375-3p promoted, whereas knockdown of miR-375-3p antagonized hypoxia-induced effects in h9c2 cells. In addition, miR-375-3p was validated to negatively regulate FOXP1 and Bcl2l2 expression through target binding, and silencing of FOXP1 and Bcl2l2 could independently abate the anti-apoptosis role of miR-375-3p knockdown in hypoxic h9c2 cells. Collectively, blocking miR-375-3p suppressed hypoxia-evoked apoptosis of cardiomyocytes by targeting and upregulating FOXP1 and Bcl2l2. Our results might suggest maternal serum miR-375-3p as a potential biomarker for prenatal detection of fetal CHD.

5-Aza-2'-deoxycytidine may enhance the frequency of T regulatory cells from CD4+ naïve T cells isolated from the peripheral blood of patients with chronic HBV infection.

OBJECTIVES: Methylation pattern of gene modification is essential for the differentiation of T regulatory cells (Tregs) and 5-Aza-2'-deoxycytidine is a common inhibitor of methylation. This study aimed to investigate the potential effects of Treg polarizing conditions and 5-Aza-2'-deoxycytidine treatment in the differentiation of naïve T cells during chronic hepatitis B virus (HBV) infection. METHODS: The frequency of Tregs in peripheral blood was determined by flow cytometry from patients with chronic hepatitis B (CHB) (n = 51), liver cirrhosis (LC) (n = 47), hepatocellular carcinoma (HCC) (n = 40) and healthy controls (HCs) (n = 17). Gene expression were detected by qRT-PCR and DNA methyltransferases (DNMT) Activity was also determined. RESULTS: The frequency of Tregs and Foxp3 expression in peripheral blood from 5-Aza-2'-deoxycytidine-treated groups were higher than that with acetic acid treatment as a control. Foxp3 mRNA and the frequency of Tregs derived from naïve CD4+T cells from peripheral blood of patients with HCC or LC were more pronounced compared with HCs. 5-Aza-2'-deoxycytidine may have induced a more pronounced upward trend of PD-1 expression in HBV patients. CONCLUSIONS: 5-Aza-2'-deoxycytidine mediated demethylation has potential effects on enhancing the differentiation of naïve T cells to Tregs in chronic HBV infection.

Characterization of immunoreactivity with whole-slide imaging and digital analysis in high-grade serous ovarian cancer.

Ovarian cancer is the most lethal of gynecological cancers with 5-year survival rate of ca. 45%. The most common histologic subtype is high-grade serous carcinoma, which typically is presented with advanced stage and development of chemoresistance. Therefore, new treatment options, including immunotherapies, are needed. Understanding the features of the immune cell populations in the tumor microenvironment is essential for developing personalized treatments and finding predictive biomarkers. Digital image analysis may enhance the accuracy and reliability of immune cell infiltration assessment in the tumor microenvironment. The aim of this study was to characterize tumor microenvironment in a retrospective cohort of high-grade serous carcinoma samples with whole-slide imaging and digital image analysis. Formalin-fixed paraffin-embedded high-grade serous carcinoma tumor tissue samples (n = 67) were analyzed for six immunohistochemical stainings: CD4, CD8, FoxP3, granzyme B, CD68, and CD163. The stained sample slides were scanned into a digital format and assessed using QuPath 0.1.2 and ImageJ software. Staining patterns were associated with clinicopathological data. The higher numbers of intraepithelial CD8+, CD163+, and granzyme B+ immune cells were associated with survival benefit when analyzed individually, while high levels of both CD8+ and granzyme B+ tumor-infiltrating lymphocytes were an independent prognostic factor in the Cox multivariate regression analysis (median progression-free survival; hazard ratio = 0.287, p = 0.002). Specimens taken after administration of neoadjuvant chemotherapy presented with lower FoxP3+ tumor-infiltrating lymphocyte density (Fisher's exact test, p = 0.013). However, none of the studied immunomarkers was associated with overall survival or clinical factors. Tumors having high amount of both intraepithelial CD8+ and granzyme B+ tumor-infiltrating lymphocytes showed better progression-free survival, possibly reflecting an activated immune state in the tumor microenvironment. The combined positivity of CD8 and granzyme B warrants further investigation with respect to predicting response to immune therapy. Neoadjuvant chemotherapy may have an effect on the tumor microenvironment and therefore on the response to immuno-oncologic or chemotherapy treatments.

Tofacitinib inhibits the development of experimental autoimmune uveitis and reduces the proportions of Th1 but not of Th17 cells.

Purpose: Tofacitinib is a pan-Janus kinase (JAK) inhibitor that suppresses cytokine signaling and in turn, the cells that participate in inflammatory immunopathogenic processes. We examined the capacity of tofacitinib to inhibit the induction of experimental autoimmune uveitis (EAU) and related immune responses. Methods: EAU was induced in B10.A mice with immunization with bovine interphotoreceptor retinoid-binding protein (IRBP), emulsified in complete Freund's adjuvant (CFA), and a simultaneous injection of pertussis toxin. Tofacitinib, 25 mg/kg, was administered daily, and the vehicle was used for control. EAU development was assessed by histological analysis of the mouse eyes, and related immune responses were assessed by (i) the levels of interferon (IFN)-γ and interleukin (IL)-17, secreted by spleen cells cultured with IRBP; (ii) flow cytometric analysis of intracellular expression by spleen, or eye-infiltrating CD4 or CD8 cells of IFN-γ, IL-17, and their transcription factors, T-bet and RORγt. In addition, the inflammation-related cell markers CD44 and CD62L and Ki67, a proliferation marker, were tested. The proportions of T-regulatory cells expressing FoxP3 were determined by flow cytometric intracellular staining, while levels of antibody to IRBP were measured with enzyme-linked immunosorbent assay (ELISA). Results: Treatment with tofacitinib significantly suppressed the development of EAU and reduced the levels of secreted IFN-γ, but not of IL-17. Further, treatment with tofacitinib reduced in the spleen and eye-infiltrating cells the intracellular expression of IFN-γ and its transcription factor T-bet. In contrast, treatment with tofacitinib had essentially no effect on the intracellular expression of IL-17 and its transcription factor, RORγt. The selective effect of tofacitinib treatment was particularly evident in the CD8 population. Treatment with tofacitinib also increased the population of CD44, but reduced the populations of cells producing CD62L and Ki67. Treatment with tofacitinib had no effect on the proportion of FoxP3 producing regulatory cells and on the antibody production to IRBP. Conclusions: Treatment with tofacitinib inhibited the development of EAU, reduced the production of IFN-γ, but had essentially no effect on the production of IL-17.

The Relationship Between Serum Concentration of Interleukin-35 and FoxP3 Polymorphism in Patients Undergoing Coronary Artery Bypass Graft Surgery.

OBJECTIVE: To investigate the association between interleukin-35 (IL-35) levels and single nucleotide polymorphisms (rs3761548, rs3761547) of the FoxP3 gene in coronary artery bypass grafting (CABG) patients. METHODS: We conducted a prospective study including 140 patients, who were scheduled for elective isolated on-pump CABG with cardiopulmonary bypass (CPB) from January 2017 to September 2018 in the Jorjani heart center. Blood samples were collected before and 12 hours after the operation. Serum levels of IL-35 were measured by enzyme-linked immunosorbent assay and the pattern of genetic variations was assessed using single specific primer-polymerase chain reaction. RESULTS: The serum concentrations of IL-35 after surgery were significantly higher than pre-surgery levels (18.4±8.3 vs. 9.89±3.2, respectively, P=0.002). There was no significant association between genotype frequencies of rs3761548 and rs3761547 and elevated IL-35 levels (P>0.05). There were significant associations between IL-35 levels and preoperative variables, including age (r=-0.34, P=0.047) and body mass index (r=-0.41, P=0.045), and intraoperative variables, including CPB time (r=0.4, P=0.02) and mean arterial pressure (r=-0.38, P=0.046), in carriers of the rs3761548 AA genotype. CONCLUSION: Serum IL-35 concentrations were significantly increased in CPB patients, which may contribute to the post-CPB compensatory anti-inflammatory response syndrome. IL-35 increased levels were not influenced by FoxP3 promoter polymorphisms (rs3761548, rs3761547).

The relationship between serum concentration of interleukin-35 and foxp3 polymorphism in patients undergoing coronary artery bypass graft surgery

Abstract Objective: To investigate the association between interleukin-35 (IL-35) levels and single nucleotide polymorphisms (rs3761548, rs3761547) of the FoxP3 gene in coronary artery bypass grafting (CABG) patients. Methods: We conducted a prospective study including 140 patients, who were scheduled for elective isolated on-pump CABG with cardiopulmonary bypass (CPB) from January 2017 to September 2018 in the Jorjani heart center. Blood samples were collected before and 12 hours after the operation. Serum levels of IL-35 were measured by enzyme-linked immunosorbent assay and the pattern of genetic variations was assessed using single specific primer-polymerase chain reaction. Results: The serum concentrations of IL-35 after surgery were significantly higher than pre-surgery levels (18.4±8.3 vs. 9.89±3.2, respectively, P=0.002). There was no significant association between genotype frequencies of rs3761548 and rs3761547 and elevated IL-35 levels (P>0.05). There were significant associations between IL-35 levels and preoperative variables, including age (r=-0.34, P=0.047) and body mass index (r=-0.41, P=0.045), and intraoperative variables, including CPB time (r=0.4, P=0.02) and mean arterial pressure (r=-0.38, P=0.046), in carriers of the rs3761548 AA genotype. Conclusion: Serum IL-35 concentrations were significantly increased in CPB patients, which may contribute to the post-CPB compensatory anti-inflammatory response syndrome. IL-35 increased levels were not influenced by FoxP3 promoter polymorphisms (rs3761548, rs3761547).

Immunoregulatory Effects of Krocina™, a Herbal Medicine Made of Crocin, on Osteoarthritis Patients: A Successful Clinical Trial in Iran.

Osteoarthritis (OA) is the major cause of joint pain and disability. This research was planned to examine the effects of Krocina™, aherbal medicine made of crocin, an ingredient of saffron, in patients with OA. Forty patients suffering from OA were enrolled in our study and randomly divided into two groups, receiving Krocina™ and placebo, and the clinical trial continued for four months.Peripheral blood was taken from all patients and the percentage ofvarious subsets of T cells in addition to the levels of forkhead box protein P3 (FOXP3) and interleukin (IL)-17 were measured by flow cytometry technique. The visualan alog scale (VAS) index analysis decreased significantly in both groups (krocinaTM and placebo) (p<0.05). Assessment of the C-reactive protein (CRP) level in serum showed a significant decrease in the krocinaTM group (p<0.05). Moreover, we found a meaningful increase in the percentage of regulatory T cells (Tregs)cellin samples gathered from Krocina™ group (P=0.02) patients. The mean percentages of T helper (Th) 17 cellsinthe Krocina™ group and CD8+ T cellsin the placebo group patients were also meaningfully reduced (p<0.05). The geometric mean fluorescence intensity (GMFI) for IL-17 showed a significant decrease and increase in Krocina™ and placebo groups, respectively (p<0.05). No noticeable difference was observed in the percentages of Th cells and GMFI-FOXP3 in either group. Treg/Th17 ratio was shifted towards Tregscell in Krocina™ group at the end of the intervention. It is concluded that Krocina™ has immunoregulatory effects on patients with OA, ameliorating the disease.

The status of FOXP3 gene methylation in pediatric systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disease caused by interaction of genetic, epigenetic, and environmental factors. One of the important epigenetic factors in SLE would be methylation of immune-related genes, such as FOXP3, which plays a role in activating the regulation and also the function of T cells. To date, the relationship between levels of serum bio-markers and the susceptibility to lupus in children has not been well-understood. In this study, the involvement of etiologic factors, such as methylation of FOXP3 gene, was investigated in children with SLE. METHOD: Twenty-four female children with SLE and 25 female healthy subjects without any history of autoimmune and inflammatory diseases were included in this study. Blood samples were obtained and DNA was extracted from the blood cells. The bisulphite method was used to convert the DNA using the MethylEdge™ Bisulfite Conversion System Kit. Then, methylation of the gene was investigated using Real Time methylation specific PCR. RESULTS: The FOXP3 DNA methylation in patients and healthy subjects was significantly different. While the median unmethylated DNA in patients was 0.57±0.43, it was 0.97±0.83 in healthy subjects (P=0.012). The Demethylation Index in patients was 0.007±0.003, significantly lower than in controls (0.014±0.013; P=0.012). CONCLUSIONS: The FOXP3 gene methylation in children with SLE was significantly higher than healthy subjects, which could possibly affect the level of gene expression. Therefore, one of the causes of increased immune response in SLE can be the lower expression of FOXP3 by hypermethylation of this gene.