Evaluation of urinary FOXP3 mRNA as a biomarker of lupus nephritis in Egyptian patients with systemic lupus erythematosus.

AIM: Lupus nephritis (LN) is one of the most serious complications of SLE. Tregs (Regulatory T lymphocytes) are thought to play a part in the pathogenesis of SLE. According to recent research, Foxp3, a Treg identification marker, plays a significant role in the pathogenesis of SLE. This study aimed to compare the urinary Foxp3 mRNA levels of patients with active and inactive forms of LN and healthy control subjects to see whether it played a role in disease activity. METHODS: We measured FOXP3 messenger RNA (mRNA) expression in the urine of 50 people with active LN, 50 people with inactive lupus, and 50 healthy people. RESULTS: We found that the expression level of FOXP3 was significantly higher in urine from patients with active LN than from subjects with inactive lupus and healthy controls (22.93 ± 4.13 vs 5.66 ± 0.47 vs 0.57 ± 0.15copy; P < 0.001).Urinary FOXP3 mRNA level significantly correlated with SLEDAI (0.000057) In the active group, urinary FOXP3 mRNA level also significantly correlated with histological activity index (< 0.00001). CONCLUSION: We concluded that urinary FOXP3 mRNA is elevated in patients with active LN and that it is linked to the SLEDAI and the severity of the disease. FOXP3 mRNA in urine sediment may be used as a non-invasive biomarker for evaluating the severity of LN and risk stratification.

Expression and Significance of MicroRNA-126 and MicroRNA-21 in Peripheral Blood Mononuclear Cells in Patients with Myasthenia Gravis.

OBJECTIVE: The aim of the study was to investigate the expression and significance of microRNA-126, microRNA-21, FOXP3mRNA, acetylcholine receptor antibody (AChR-Ab), and interleukin 6 (IL-6) in peripheral blood of patients with myasthenia gravis (MG). METHODS: From September 2015 to March 2018, 60 patients with MG who were first diagnosed as MG were selected as the MG group, and 50 healthy people in the same period were selected as the normal group. RT-PCR technology was used to detect the relative expression of microRNA-126, microRNA-21, and FOXP3mRNA in peripheral blood mononuclear cells of the MG group and the control group. The EILISA and IPA technique was used to detect the expression levels of IL-6 and AChR-Ab in the peripheral serum of the 2 groups. RESULTS: There were no differences between groups regarding patient's characteristics and baseline data. microRNA-21 and microRNA-126 are highly conserved in the human genome. microRNA-21, microRNA-126, FOXP3mRNA, AChR-Ab, and IL-6 are differentially expressed in the MG group and the control group (p < 0.05). microRNA-21 is positively correlated with AChR-Ab and IL-6 in MG patients (r = 0.746, 0.789, p < 0.05), but has no correlation with FOXP3mRNA (r = -0.249 p = 0.055), while micro-RNA-126 is positively correlated with FOXP3mRNA and negatively correlated with AChR-Ab and IL-6 (r = 0.526, -0.797, -0.801, p < 0.05). CONCLUSION: Patients with MG have differential expression of microRNA-21 and microRNA-126 and may participate in the pathogenesis of MG by regulating pathways related to inflammatory immune response.

Efficacy of Loulu Shengma Tang Combined with Azithromycin in Treating Children with Mycoplasma Pneumonia with Obstruction of Lung by Pathogenic Heat and Its Effect on Treg and Foxp3 mRNA / 中国实验方剂学杂志

Objective:To investigate the clinical efficacy of Loulu Shengma Tang combined with azithromycin in the treatment of pediatric mycoplasma pneumoniae pneumonia（MPP） with obstruction of lung by pathogenic heat， and its effect on inflammatory factors， treg and Foxp3 mRNA. Method:Totally 274 children with MPP were divided into observation group （137 cases） and control group （137 cases）. Observation group was treated with Loulu Shengma Tang combined with azithromycin dry suspension， while control group was treated with azithromycin dry suspension alone. The changes of traditional Chinese medicine（TCM） syndrome score of pathogenic-heat obstruction in the lung， serum inflammatory cytokines ［interleukin-6 （IL-6），interleukin-10 （IL-10），tumor necrosis factor-α （TNF-α），C-reactive protein （CRP）］， CD4+CD25+Treg， CD4+Foxp3+Treg and Foxp3 mRNA expressions were observed after treatment. The clinical efficacy and the incidence of adverse reactions were compared between two groups. Result:The total effective rate of observation group was 94.16%（129/137） after treatment， which was significantly higher than 77.37% （106/137）of observation group （P<0.05）. The disappearance times of cough， lung rale， fever and lung shadow in observation group were shorter than that in control group （P<0.05）. After treatment， TCM syndrome score of pathogenic-heat obstruction in lung was significantly higher than those in control group （P<0.05）， serum IL-6， IL-10， TNF-α and CRP levels in observation group were significantly lower than those in control group （P<0.05， P<0.01）， while CD4+CD25+Treg， CD4+Foxp3+Treg and Foxp3 mRNA expressions were significantly higher than those in control group （P<0.05）. The incidence of adverse reactions in observation group was 7/137 （5.11%）， which was significantly lower than 16/137 （11.68%） in control group. Conclusion:The clinical efficacy of Loulu Shengma Tang combined with azithromycin dry suspension in the treatment of pediatric MPP and its effect on serum inflammatory factors （IL-6， IL-10， TNF-α， CRP）， regulatory T cells and Foxp3 mRNA expressions were better than those of azithromycin dry suspension alone. The incidence of adverse reactions of Loulu Shengma Tang was lower than that of azithromycin dry suspension alone.

Genome-wide gene expression analysis for target genes to differentiate patients with intestinal tuberculosis and Crohn's disease and discriminative value of FOXP3 mRNA expression.

BACKGROUND AND AIMS: Crohn's disease (CD) and intestinal tuberculosis (ITB) are both chronic granulomatous conditions with similar phenotypic presentations. Hence, there is need for a biomarker to differentiate between both these two diseases. This study aimed at genome-wide gene expression analysis of colonic biopsies from confirmed cases of ITB and CD in comparison with controls. To evaluate the role of T regulatory cells, forkhead box P3 (FOXP3) mRNA expression was quantified in serum as well as in colonic biopsies from patients with ITB and with the controls. METHODS: Paired samples, including serum and colonic biopsies, were taken from 33 study subjects (CD, ITB and controls), and total RNA was extracted. Human whole genome gene expression microarray analysis was performed using the Illumina HumanWG-6 BeadChip Kit with six total RNA samples of the three groups in duplicates. Real-time PCR for FOXP3 mRNA expression was analyzed in serum samples and colonic biopsy samples (4-CD, 5-ITB, 4-controls). RESULTS: In CD and ITB there was 1.5-fold upregulation of 92 and 382 genes and 1.5-fold downregulation of 91 and 256 genes, respectively. Peroxisome proliferators via the PPARγ pathway were most significantly downregulated (P < 0.005) in CD. Additionally, the IL4/5/6 signaling pathways and Toll-like receptor signaling pathway were identified as significantly differentially regulated (P < 0.005) at > 2-fold change. In ITB, the complement activation pathway, specifically the classical pathway, was the most significantly upregulated. FOXP3 mRNA expression was significantly elevated in colonic biopsies obtained from ITB patients as compared with CD cases (4.70 ± 2.21 vs 1.48 ± 0.31, P = 0.016). CONCLUSIONS: FOXP3 mRNA expression in colonic mucosa could be a discriminatory marker between ITB and CD. Upregulation of the complement activation pathway in ITB suggests that pathogenetic mechanisms for ITB are similar to those of pulmonary tuberculosis. In CD, downregulation of PPARγ was seen in colonic tissue, suggesting that restoration of PPARγ-dependent anti-microbial barrier function may be a therapeutic target.

Role for urinary biomarkers in diagnosis of acute rejection in the transplanted kidney.

Despite the introduction of potent immunosuppressive medications within recent decades, acute rejection still accounts for up to 12% of all graft losses, and is generally associated with an increased risk of late graft failure. Current detection of acute rejection relies on frequent monitoring of the serum creatinine followed by a diagnostic renal biopsy. This strategy is flawed since an alteration in the serum creatinine is a late clinical event and significant irreversible histologic damage has often already occurred. Furthermore, biopsies are invasive procedures that carry their own inherent risk. The discovery of non-invasive urinary biomarkers to help diagnose acute rejection has been the subject of a significant amount of investigation. We review the literature on urinary biomarkers here, focusing on specific markers perforin and granzyme B mRNAs, FOXP3 mRNA, CXCL9/CXCL10 and miRNAs. These and other biomarkers are not yet widely used in clinical settings, but our review of the literature suggests that biomarkers may correlate with biopsy findings and provide an important early indicator of rejection, allowing more rapid treatment and better graft survival.

Suppressive activity and altered conventional phenotype markers/mediators of regulatory T cells in patients with self-limiting hepatitis E.

Hepatitis E virus (HEV) is a major cause of self-limiting acute viral hepatitis in several developing countries. Elevated levels of peripheral CD4(+) CD25(+) Foxp3(+) , CD4(+) CD25(-) Foxp3(+) and rise in IL-10 in hepatitis E have been associated with the involvement of regulatory T cells (Treg). The functional role of the same is yet elusive. In the current study, we have assessed (i) Foxp3 expression by real-time PCR and by flow cytometry, (ii) the levels of antigen-specific IL-10 and TGF-ß by ELISA, (iii) functional analysis of Treg cells and (iv) expression of Treg-associated conventional phenotypes by flow cytometry in 54 acute patients, 44 recovered individuals from hepatitis E and in 33 healthy controls. Foxp3 mRNA elevation in the acute compared with recovered group and elevation in Foxp3(+) cells in both patient groups were significantly elevated. The levels of IL-10 and TGF-ß in the acute patients and TGF-ß in the recovered individuals were elevated. Significantly higher expression of CTLA-4, PD1, GITR, CD95, CD103 and CD73 on Treg and T effector (Teff) cells was detected in the patient groups. Treg cells of acute patients and recovered individuals exhibited suppressive activity indicating that the Treg cells of hepatitis E patients are functional. The suppressive capacity of Treg cells in acute hepatitis E patients was significantly higher compared with the recovered individuals. Based on our findings, the suppressive functionality of these key markers associated with hepatitis E Treg function need further exploration to get a better understanding of the mechanisms of Treg-mediated suppression.

Up-regulate of the proportion of CD4+CD25+ regulatory T cells by lymphoma cell EL-4 supernatants / 肿瘤研究与临床

Objective To explore if the tumor cell can up-regulate the proliferation of CD4+CD25+ Treg cells. Methods Lymphoma cell EL-4 supernatant was combined with the normal mise spleen lymphocyte. After cultured 72 hours, CD4+CD25+ subset were analyzed by flow cytometry and the gene expression level of the specific marker Foxp3 were tested by RT-PCR. The experiment repeated three times. Results The supematant derived from EL-4 can increase the proportion of CD4+CD25+ Treg cells in normal mouse spleen lymphocytes than that of the control group. Moreover, when added the CD3 McAb with EL-4 supematant together, the number of CD4+CD25+ Treg cells increased. The level of corresponding Foxp3 mRNA also increased. Conclusion These data suggested that in the supernatant may occur immune regulatory factors which up-regulated the number of CD4+CD25+ Treg cells and indicated that the tumorigenesis can facilitate proliferation of CD4+CD25+ Treg cells.

Significance of the detections for CD4 +CD25 + regulatory T cells, Foxp3 mRNA and interleukin 2 receptor in kidney transplantation recipients / 中华肾脏病杂志

Objective To observe the changes of CD4+CD25+ regulatory T cells, Foxp3 mRNA and soluble interlukin 2 receptor (sIL-2R) in the peripheral blood of kidney transplantation recipients and to evaluate their effect on the diagnosis of acute rejection. Methods Forty-two renal transplant recipients and 30 healthy controls were enrolled in this study. CD4+CD25+ regulatory T cells proportion, Foxp3 mRNA and sIL-2R of pre-transplantation and those of day 7,14, 28, 56 of post-transplantation were measured by flow cytometer, fluorescent quantization PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Biochemistry appliance was used to detect serum creatinine. The diagnosis of acute rejection in transplanted kidney was based on the clinical symptoms, the laboratory examinations, Doppler ultrasound and biopsy. Results (1)At day 7, 14, 28, 56 of post-transplantation, CD4+CD25+ regulatory T ceils proportion, Foxp3 mRNA level in acute rejection group were significantly decreased compared with those in non-acute rejection group. (2) There were significant differences of peripheral blood CD4+CD25+ regulatory Tcells[(9.22±3.53)% vs (6.09±1.99)%, P<0.01], Foxp3 mRNA[(0.82±0.36)×10-3 vs (0.50±0.28)×10-3, P<0.01] and sIL-2R levels [(856.30±108,24) U/ml vs (247.35±11.24) U/ml, P<0.01]between patients of pre-transplantation and healthy control group. (3)Plasma CD4+CD25+ regulatory T cells [(16.53±4.14)%] and the expression of Foxp3 mRNA [(4.97±1.94)×10-3] was significantly increased, but sIL-2R level [(463.72±31.23) U/ml] was significantly decreased as the transplanted renal function was restored (all P<0.01). (4) Plasma CD4+CD25+regulatory T cells [(12.18~2.86)%] and the expression of Foxp3 mRNA [(3.15±1.22)×10-3] was significantly decreased (P<0.01), and sIL-2R level [(748.36±115.41) U/ml] was significantly increased (P<0.01) when acute rejection occurred. The above changes had an earlier onset than the change of Scr. (5)The percentage of CD4+CD25+ regulatory T cells was positively correlated with the Foxp3 mRNA level (P<0.01), but was not correlated with sIL-2R level in all the patients. Conclusion The measurement of these markers in peripheral blood may be an important guideline to the diagnosis and prognosis of acute rejection in renal transplant recipients.

Analysis of CD4+CD25+ Regulatory T Cells and Foxp3 mRNA in the Peripheral Blood of Patients with Asthma / 华中科技大学学报（医学）（英德文版）

The changes of CD4+CD25+ regulatory T cells (CD4+CD25+ Treg) and Foxp3 mRNA in peripheral blood mononuclear cells (PBMCs) from patients with asthma were investigated in order to elucidate the possible roles of CD4+CD25+ Treg in the development of asthma. The peripheral blood samples were collected from 29 healthy controls (normal control group) and 78 patients with asthma which included 30 patients in exacerbation group, 25 patients in persistent group, and 23 patients in remission group. By using flow cytometry and RT-PCR, the CD4+CD25+ Treg ratio and Foxp3 mRNA in PBMCs were detected. The CD4+CD25+ Treg ratio and Foxp3 mRNA in PBMCs of exacerbation and persistent groups were lower than that of remission and normal control groups (P＜0.05). Although the CD4+CD25+ Treg ratio and Foxp3 mRNA of remission group were also lower than that of normal control group, there was no significant difference between them (P＞0.05). As compared with persistent group, exacerbation group had lower CD4+CD25+ Treg ratio and Foxp3 mRNA (P＜0.05). It was indicated that the decrease of CD4+CD25+Treg ratio and its function in PBMCs may be responsible for pathogenesis of asthma.