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BackgroundA previous validation study showed a very low sensitivity and higher specificity associated with Hanifin and Rajka criteria (H&R) and the UK Working Party criteria (UKWP) in diagnosing AD vs. the Chinese criteria of atopic dermatitis (AD) for children (CCAD). However, their diagnostic efficacy in adult and elderly Chinese populations remains unknown.ObjectivesTo validate the diagnostic efficacy of three sets of AD criteria in adult and elderly Chinese populations in a hospital setting.MethodsA total of 1034 patients (aged 19–95 years) from five university hospital dermatological clinics were recruited. Medical history, dermatological examination, AD diagnosis and evaluation of AD severity were done by dermatologists. Each patient was investigated by two dermatologist panels, one to establish a clinical diagnosis, and the other to identify and record the major or minor signs of H&R criteria, UKWP criteria and CCAD. Taking clinical diagnosis as the reference, the diagnostic efficacy of three sets of diagnostic criteria was evaluated. The χ2 test or rank sum test were used for between-groups comparisons.ResultsCCAD had a higher sensitivity (84.0%), especially among mild and moderate cases of AD (72.7% and 90.3%, respectively), than the H&R (58.0%; P < 0.001) and UKWP criteria (56.0%; P < 0.001) in diagnosing AD. The specificity of CCAD (92.7%) was slightly lower than the H&R (97.3%; P < 0.001) or UKWP criteria (97.4%; P < 0.001). The CCAD had the highest Youden index (0.77), accuracy rate (0.90) and Kappa value (0.76) of the three sets of diagnostic criteria.ConclusionsConsistent with results in a population of Chinese children, although the H&R and UKWP criteria had a high specificity for diagnosing AD, their low sensitivity limited their use in adult and elderly Chinese patients. Based on the high sensitivity and favourable diagnostic efficacy, the CCAD is proposed for AD diagnosis in adult and elderly Chinese populations, especially for cases of mild and moderate AD.

Atopic dermatitis (AD) is a chronic, pruritic, inflammatory skin disease with rising prevalence. Topical corticosteroids (TCS) are recommended as first-line therapy for patients with AD in China; however, corticophobia is a widespread concern, which can manifest as noncompliance: in a previous Chinese study, almost all parents whose children had AD were very concerned about the side effects of TCS and, as a result, nearly half did not use it in the event of recurrence. We propose a TCS-sparing treatment algorithm for the management of infants, children, adolescents, and adults with mild-to-moderate AD, to guide clinical practice in China.A panel of eight experts in AD from China and one expert from Germany formed to develop a practical algorithm for the management of mild-to-moderate AD, focusing on pimecrolimus.Irrespective of body location, all patients with mild AD (including acute flares) and infants with moderate AD should apply the topical calcineurin inhibitor (TCI) pimecrolimus twice daily to the affected area until symptoms disappear. For children, adolescents, and adults with moderate AD, pimecrolimus should be applied twice daily to sensitive skin areas, and a TCI (either pimecrolimus or tacrolimus) should be applied twice daily to other body locations. Short-term administration of TCS, followed by TCI twice daily, is recommended for most patients with moderate AD experiencing acute flares, regardless of lesion site. Emollients should be used regularly.The algorithm presented intends to simplify treatment of AD in China and guide clinical decision-making.

Background: Anxiety and depression in hypertensive patients may lead to poor blood pressure control and increases the risk of disease mortality. Lifestyle impacts depression and anxiety. This study aimed to assess cardiovascular health behaviors (CHBs) associated with co-morbid anxiety and depression among patients with hypertension.Methods: We included 488 hypertensive patients from Changsha, China. We measured anxiety and depression using the self-rating anxiety scale (SAS), and the self-rating depression scale (SDS), respectively. CHBs (smoking, overweight/obesity, leisure physical activity (LPA), and fruit and vegetable consumption (FVC)) were assessed using the Cardiovascular Health in Ambulatory Care Research Team (CANHEART) index. We compared anxiety and depression prevalence of participants in different socio-demographic, CHBs, co-morbidities, family histories, assessed risk factors and their biological synergistic interaction.Results: The proportion of not ideal in smoking, overweight/obesity, LPA, and FVC were 63.9%, 49.8%, 30.7%, 78.3%, respectively. Displaying comorbidity of HLP and not obtaining adequate amounts of LPA were risk factors for both anxiety and depression. The synergy interaction between obtaining secondary education or less and displaying comorbidity of hyperlipidemia (HLP) for anxiety (OR=5.238, 95% CI=2.784, 9.856), and between not obtaining adequate amounts of LPA and obtaining manual labor for depression (OR=7.164, 95% CI=3.553, 14.443) was statistically significant.Conclusion: Our study indicated that doctors should pay more attention to the psychiatric health of hypertensive patients at lower education levels and with second comorbidities. More importantly, patients with hypertension working on manual jobs need to pay closer attention to how they spend their leisure time and strengthen LPA.

Background: Anxiety and depression in hypertensive patients may lead to poor blood pressure control and increases the risk of disease mortality. Lifestyle impacts depression and anxiety. This study aimed to assess cardiovascular health behaviors (CHBs) associated with co-morbid anxiety and depression among patients with hypertension.Methods: We included 488 hypertensive patients from Changsha, China. We measured anxiety and depression using the self-rating anxiety scale (SAS), and the self-rating depression scale (SDS), respectively. CHBs (smoking, overweight/obesity, leisure physical activity (LPA), and fruit and vegetable consumption (FVC)) were assessed using the Cardiovascular Health in Ambulatory Care Research Team (CANHEART) index. We compared anxiety and depression prevalence of participants in different socio-demographic, CHBs, co-morbidities, family histories, assessed risk factors and their biological synergistic interaction.Results: The proportion of not ideal in smoking, overweight/obesity, LPA, and FVC were 63.9%, 49.8%, 30.7%, 78.3%, respectively. Displaying comorbidity of HLP and not obtaining adequate amounts of LPA were risk factors for both anxiety and depression. The synergy interaction between obtaining secondary education or less and displaying comorbidity of hyperlipidemia (HLP) for anxiety (OR=5.238, 95% CI=2.784, 9.856), and between not obtaining adequate amounts of LPA and obtaining manual labor for depression (OR=7.164, 95% CI=3.553, 14.443) was statistically significant.Conclusion: Our study indicated that doctors should pay more attention to the psychiatric health of hypertensive patients at lower education levels and with second comorbidities. More importantly, patients with hypertension working on manual jobs need to pay closer attention to how they spend their leisure time and strengthen LPA.

e21502 Background: Exciting progress has been recently witnessed on the development of chimeric antigen receipt T cell (CAR-T) therapy for hematopoietic malignancies. The biological principles of CAR-T therapy are applicable to solid tumors but very little success has been achieved due to various challenges intrinsic to solid tumors. The development of new solid tumor targets is particularly important. CD276 (B7H3) is one of the most promising therapeutic targets for solid tumors and has been extensively tested in preclinical trials. However, no clinical trials have been reported on its efficacy and safety. In this study, we report a case of relapsed basal cell carcinoma (BCC) patient treated with B7H3 CAR-T cells through direct intra-tumor injection. Trial registration number: ChiCTR2100044386. Methods: We have generated a 2nd generation of B7H3 CAR-T, the extracellular scFv was derived from a humanized B7H3 monoclonal antibody, which was generated by hybridoma technology. Autologous CAR-T cells were manufactured in a cGMP facility. CAR-T cells were given by three dose-escalated intra-tumor injections without lymphodepletion conditioning. Results: A 65-year-old man was initially diagnosed with multiple BCC, the disease relapsed and spread to all over the body. Immunohistochemical staining of the tumor tissue showed positive for B7H3 (80%, ++̃+++). The patient received autologous anti-B7H3 CAR-T therapy cells through intra-tumor injection three times with dose escalation on day 1, day 14 and day 62. There were two different tumor sites that were treated in this patient (site 1 for abdominal tumor 1.8 cm × 0.6 cm; site 2 for forehead tumor 6 cm × 6 cm). For the adverse events, the patient’s AST/ALT levels slightly increased about two fold after the first infusion. However, we did not observe increases of AST/ALT levels after the second and third infusion, even with much higher dose. After three infusions, the volume of original lesion in the abdomen decreased 40% (1.8cm × 0.6cm to 1.3cm × 0.5cm), shrinking from bulging to flat. The large lesion in the forehead became dry from original ulcer and bleeding. We further performed surgery to remove the remained tumor in the abdomen after the third infusion. IHC analysis demonstrated that the percentage of B7H3 positive cells decreased from 80% to about 40%, and the intensity also decreased from ++̃+++ to +. Meanwhile, CD3 T cells increased in the tumor site, but failed to efficiently infiltrate into neighboring tumor areas, which was marked by high level of TGF-β. Conclusions: In this case, we reported for the first time that a patient with skin tumor achieved a partial response after receiving repeated intra-tumor injections of anti-B7H3 CAR-T cells. This study demonstrated that B7-H3 CAR-T cell therapy is a safe and promising treatment for relapsed skin tumor. Combination with other strategies to improve CAR-T cell infiltration in the tumor site may help to further improve the clinical response. Clinical trial information: ChiCTR2100044386.

Pemphigus vulgaris is an autoimmune blistering disease characterized by a loss of epidermal cell–cell adhesion caused by anti-desmoglein (Dsg) autoantibodies. The pathogenesis of PV remains unclear. However, abnormal frequency and function of Th2 cells are believed to contribute to PV. We investigated Nr4a2, a transcription factor, which has been found to regulate T cell differentiation, for its association with Th2 cell differentiation and functions in PV. We found significantly decreased mRNA and protein levels of Nr4a2 in CD4+ T cells from patients with PV, compared with healthy control subjects. In addition, mRNA and protein levels of Nr4a2 in CD4+ T cells were inversely correlated with serum levels of IL-4 and IL-13 in patients with PV. Overexpression of Nr4a2 in CD4+ T cells from patients with PV significantly reduced the mRNA levels of GATA3, IL-4, and IL-13, while Nr4a2 siRNA treatment showed the reverse effects on the expression of these Th2-related cytokines and transcription factors. The data suggest that the altered level of Nr4a2 in CD4+ T cells is associated with the development of PV. Nr4a2 may contribute to the pathogenesis of PV by negatively regulating Th2 activity and secretion of Th2-related cytokines.

Background In patients with hormone receptor-positive postmenopausal of early stage breast cancer, adjuvant endocrine monotherapies include letrozole, anastrozole, exemestane, toremifene and tamoxifen. But the optimum regimen remains controversial. Methods PubMed, Cochrane Database and ClinicalTrials.gov were systematically reviewed of abstract for randomized-controlled trials (RCTs) to assess the efficacy of tamoxifen, letrozole, exemestane, anastrozle and toremifene for postmenopausal patients with hormone-receptor positive (HR+), who have not received prior therapy for early stage breast cancer. The outcomes were measured by disease-free survival (DFS) and overall survival (OS). We evaluated relative hazard ratios (HRs) for death of different therapies by combination hazard ratios for death of included trials. The SUCRA values were used to evaluate the rankings of efficacy for these monotherapies. Results A total of fourteen studies including 19,517 patients in our research were absorbed and estimated. The superiority of efficacy for DFS were 5-year letrozole and 10-year tamoxifen (SUCRA values 0.743/0.657) in all comparisons. A more efficient SUCRA values for OS were 5-year Exemestane, 5-year letrozole and 10-year tamoxifen (0.756/0.677/0.669). Conclusions Clinically important differences exist between commonly prescribed different adjuvant endocrine monotherapy regimens for both efficacy and acceptability in favor of exemestane and letrozole. 10-year tamoxifen for early breast cancer patients is noninferior to 5-year anastrozle, and might be the best choice where aromatase inhibitors (AIs) are not easy to acquire.

Fc receptor γ subunit (FcRγ)-related receptors expressed on antigen-presenting cells (APCs) enhance allergen sensitization and allergic inflammation. DNA demethylation of the high-affinity IgE receptor γ subunit gene (FCER1G) leads to FcRγ and FcεRI overexpression on monocytes from patients with atopic dermatitis.We investigated epigenetic mechanisms underlying FCER1G demethylation and upregulation of FcRγ-related receptors on APCs and the consequent effect on allergic responses.Effects of thymic stromal lymphopoietin (TSLP) on expression of FcRγ and its related receptors and methylation or hydroxymethylation of FCER1G in human monocytes were assessed. Recruitment of ten-eleven translocation protein (TET) 2 to FCER1G by TSLP-activated phosphorylated signal transducer and activator of transcription 5 (pSTAT5) was evaluated. Effects of TSLP on expression of FcRγ-related receptors and costimulatory receptors on monocyte-derived dendritic cells (DCs) and the ability of DCs to take up ovalbumin were analyzed. TSLP-induced TpSTAT5 activation by TSLP resulted in TET2 recruitment to FCER1G, leading to FCER1G demethylation and subsequent upregulation of FcRγ-related receptors on monocytes. TSLP not only stimulated monocyte-derived DC maturation but also maintained their allergen uptake ability, likely through maintenance and upregulation of FcRγ-related receptors. Allergen sensitization and upregulation of TTSLP mediated upregulation of FcRγ-related receptors on APCs through activation of pSTAT5, which recruited TET2 to induce FCER1G demethylation. TSLP-induced allergic T

To illuminate a method for establishment of a cost-efficient atopic dermatitis (AD) mouse model by topical application of ovalbumin (OVA), super-antigen staphylococcal enterotoxin B (SEB), and calcipotriene ointment (CO) on the back of BALB/c mice. Methods: Experimental mice were topically treated with OVA/SEB or OVA/SEB/CO every other day during 15 days of induction. Clinical alterations on the skin area were monitored every other day. Epidermal thickness were measured by reflectance confocal microscope (RCM) before harvest. Inflammatory cells in skin biopsies were marked by hematoxylin-eosin (HE) staining. Blood sample and skin biopsies were measured by ELISA and quantitative real-time PCR to detect the expression of IL-2, IL-4, IL-31, interferon (IFN)-γ, tumor necrosis factor (TNF)-α pruritus-associated nerve growth factor (NGF), and serum IgE. Results: Human AD-like cutaneous local inflammatory reaction was characterized by the accumulation of inflammatory cells, increased epidermal thickness and serum IgE levels as well as Th1 cell-associated cytokines (IFN-γ, TNF-α), Th2 cell-associated cytokines (IL-4, IL-31), and NGF in the OVA/SEB/CO group compared with that in the normal control group or the OVA/SEB group. Conclusion: OVA/SEB/CO can induce an AD-like mouse model with lower economic and time consumption.目的：阐明利用卵清蛋白(ovalbumin，OVA)、金黄色葡萄球菌肠毒素B(staphylococcal enterotoxin B，SEB)和维生素D类似物(calcipotriene ointment，CO)局部刺激小鼠背部建立特应性皮炎(atopic dermatitis，AD)动物模型的方法。方法：将BALB/c小鼠背部去毛随机分为3组：对照组，OVA/SEB，OVA/SEB/CO，分别给予相应的处理。隔天给药并拍照记录皮损状态，于第15天处死小鼠，处死前用皮肤共聚焦显微镜测量表皮厚度。用ELISA方法检测血清IL-2，IL-4，IL-31，干扰素(interferon，IFN)-γ，肿瘤坏死因子(tumor necrosis factor，TNF)-α，神经生长因子(nerve growth factor，NGF)和IgE水平，HE染色检测皮损处炎症情况，实时荧光定量PCR检测皮损处上述因子的mRNA表达水平。结果：OVA/SEB/CO组皮损处炎症细胞浸润和表皮增厚明显，血清IgE水平显著增高，血清和皮损处IL-4，IL-31，IFN-γ，TNF-α和NGF表达水平均显著高于对照组或OVA/SEB组。结论：OVA/SEB/CO局部刺激可短时间、低成本地诱导BALB/c小鼠皮肤AD样改变。.

Background Fc receptor γ subunit (FcRγ)-related receptors expressed on antigen-presenting cells (APCs) enhance allergen sensitization and allergic inflammation. DNA demethylation of the high-affinity IgE receptor γ subunit gene (FCER1G) leads to FcRγ and FceRI overexpression on monocytes from patients with atopic dermatitis. Objective We investigated epigenetic mechanisms underlying FCER1G demethylation and upregulation of FcRγ-related receptors on APCs and the consequent effect on allergic responses. Methods Effects of thymic stromal lymphopoietin (TSLP) on expression of FcRγ and its related receptors and methylation or hydroxymethylation of FCER1G in human monocytes were assessed. Recruitment of ten-eleven translocation protein (TET) 2 to FCER1G by TSLP-activated phosphorylated signal transducer and activator of transcription 5 (pSTAT5) was evaluated. Effects of TSLP on expression of FcRγ-related receptors and costimulatory receptors on monocyte-derived dendritic cells (DCs) and the ability of DCs to take up ovalbumin were analyzed. TSLP-induced TH polarization and related cytokine production were also analyzed. Results pSTAT5 activation by TSLP resulted in TET2 recruitment to FCER1G, leading to FCER1G demethylation and subsequent upregulation of FcRγ-related receptors on monocytes. TSLP not only stimulated monocyte-derived DC maturation but also maintained their allergen uptake ability, likely through maintenance and upregulation of FcRγ-related receptors. Allergen sensitization and upregulation of TH2/TH17-related cytokines contributed to TSLP-DC–induced TH2/TH17 polarization. The latter was attenuated on neutralization with a dectin-2 antibody. Conclusions TSLP mediated upregulation of FcRγ-related receptors on APCs through activation of pSTAT5, which recruited TET2 to induce FCER1G demethylation. TSLP-induced allergic TH2/TH17 polarization likely depends on dectin-2–mediated allergen sensitization and upregulation of TH2/TH17-related cytokines.

To cite this article: Liang Y, Wang P, Zhao M, Liang G, Yin H, Zhang G, Wen H, Lu Q. Demethylation of the FCER1G promoter leads to FceRI overexpression on monocytes of patients with atopic dermatitis. Allergy 2012; 67: 424–430. Abstract Background: Overexpression of the high-affinity receptor for immunoglobulin E on atopic monocytes and dendritic cells is known to contribute to the pathogenesis of atopic dermatitis (AD). However, it remains unclear what is the underlying mechanism of FceRI deregulation. It has been speculated that epigenetic deregulation may play a role. Methods: Global DNA methylation levels of monocytes from 10 AD patients and 10 healthy controls were measured using a global DNA methylation kit. Bisulfite sequencing was performed to determine the methylation status of the FCER1G promoter region. FceRIγ mRNA and FceRI protein levels were detected by real-time RT-PCR, Western blotting, and flow cytometry, respectively. Patch methylation and the demethylating agent, 5-azacytidine, were used to determine the functional significance of methylation changes on FceRI expression. Results: Monocytes from AD patients show a global hypomethylation, as well as a locus-specific hypomethylation at FCER1G promoter, as compared to healthy controls. Furthermore, this hypomethylation of FCER1G is inversely correlated with its expression. Patch methylation in combination with luciferase reporter assay confirmed the direct relationship between methylation and expression. Moreover, treating healthy monocytes with 5-azacytidine caused a reduction in methylation levels and an induction in FceRIγ transcription and surface expression of FceRI. Conclusion: Demethylation of specific regulatory elements within the FCER1G locus contributes to FceRI overexpression on monocytes from patients with AD.

MicroRNAs (miRNAs) consist of a class of evolutionarily conserved small non-coding RNA that regulates target messenger RNAs by mechanisms such as incomplete base pairing and post-transcriptional gene silencing. Recent studies have shown that aberrant miRNA expression is a common feature of many human disorders including aging, heart diseases, cancer, autoimmune diseases and others. It seems likely that miRNA expression levels can be used as novel diagnostic markers. MiRNAs may also provide a new strategy for therapeutic interventions. In this review, we focus on recent advances in understanding how miRNA changes contribute to disease development and their potential as novel biomarkers and therapeutic targets for human diseases.

Atopic dermatitis (AD) is a chronic inflammatory skin disease caused by a combination of genetic and environmental factors. Genetic evidences depict a complex network comprising by epidermal barrier dysfunctions and dysregulation of innate and adaptive immunity in the pathogenesis of AD. Mutations in the human filaggrin gene (FLG) are the most significant and well-replicated genetic mutation associated with AD, and other mutations associated with epidermal barriers such as SPINK5, FLG-2, SPRR3, and CLDN1 have all been linked to AD. Gene variants may also contribute to the abnormal innate and adaptive responses found in AD, including mutations in PRRs and AMPs, TSLP and TSLPR, IL-1 family cytokines and receptors genes, vitamin D pathway genes, FCER1A, and Th2 and other cytokines genes. GWAS and Immunochip analysis have identified a total of 19 susceptibility loci for AD. Candidate genes at these susceptibility loci identified by GWAS and Immunochip analysis also suggest roles for epidermal barrier functions, innate and adaptive immunity, interleukin-1 family signaling, regulatory T cells, the vitamin D pathway, and the nerve growth factor pathway in the pathogenesis of AD. Increasing evidences show the modern lifestyle (i.e., the hygiene hypothesis, Western diet) and other environmental factors such as pollution and environmental tobacco smoke (ETS) lead to the increasing prevalence of AD with the development of industrialization. Epigenetic alterations in response to these environmental factors, including DNA methylation and microRNA related to immune system and skin barriers, have been found to contribute to the pathogenesis of AD. Genetic variants and epigenetic alteration might be the key tools for the molecular taxonomy of AD and provide the background for the personalized management.

Background The pathogenesis of systemic lupus erythematosus (SLE) has not yet been completely elucidated. One of the hallmarks of SLE is the production of autoantibodies by uncontrolled over-activated B cells. Early B cell factor 1 (EBF1) contributes to the development, activation, and proliferation of B cells through activation of the AKT signaling pathway. Accumulating evidence has demonstrated that several microRNAs (miRNAs) contribute to the pathogenesis of autoimmune diseases through the regulation of B cells in SLE. We aim to investigate the expression patterns of miR-1246 in B cells and its contribution to pathogenesis of SLE. Results Our results showed that the expression of miR-1246 was significantly decreased in B cells from SLE patients. We verified that miR-1246 specifically targeted the EBF1 messenger RNA (mRNA) by interacting with its 3′-untranslated region (3′-UTR) and regulated the expression of EBF1. Transfection of miR-1246 inhibitors into healthy B cells upregulated the expression of EBF1, enhanced B cell function, and increased the production of B cell surface co-stimulatory molecules CD40, CD80, and CD86. We also observed that abnormal activation of the AKT signaling pathway was associated with decreased P53 expression, leading to the downregulation of the miR-1246 expression; and upregulation of the miR-1246 expression reversed the responsiveness of B cells by inhibiting EBF1 expression. Conclusions Activated B cells in lupus could decrease the expression of miR-1246 through the AKT-P53 signaling pathway, which in turn enhances the expression of EBF1, thereby promoting further activation of B cells. Conversely, upregulation of miR-1246 could interrupt this amplification pathway. Our findings thus provide a theoretical framework towards the research of novel biological targets in SLE treatment. Electronic supplementary material The online version of this article (doi:10.1186/s13148-015-0063-7) contains supplementary material, which is available to authorized users.

Summary Background Systemic sclerosis (SSc) is characterized by an excessive production of extracellular matrix. It is widely accepted that fibrosis is induced by transforming growth factor (TGF)-β in the early stage and is subsequently maintained by connective tissue growth factor (CTGF). CTGF is a cysteine-rich mitogenic peptide that has been involved in various fibrotic disorders and can be induced in fibroblasts by activation with TGF-β. Objectives To evaluate the effect of small interfering RNA (siRNA) targeting CTGF on the expression of CTGF and type I and type III collagen in SSc. Methods Skin fibroblasts from patients with SSc were cultured in vitro and later transfected using four CTGF-specific siRNAs and one nonspecific siRNA. The effect of CTGF-specific siRNAs on the expression of CTGF and type I and type III collagen was examined and quantified by real-time reverse transcription–polymerase chain reaction (RT–PCR), Western blot analysis and immunocytochemistry. Results Semiquantitative RT–PCR analysis showed that the four CTGF-specific siRNAs significantly reduced CTGF mRNA expression (P

To explore the mechanisms by which DNA methylation regulates miR-126 and its host gene EGFL7 in CD4+ T cells from patients with systemic lupus erythematosus (SLE).We analyzed the expression and the DNA methylation status within promoter region of EGFL7 and miR-126 by real-time qPCR and bisulfite genomic sequencing analysis.miR-126 and EGFL7 mRNA expression was upregulated in CD4+ T cells from SLE compared with that from healthy controls (P0.01). EGFL7 mRNA level was positively correlated with miR-126 expression in CD4+ T cells from SLE (r=0.538, P=0.015). The average methylation level of EGFL7 promoter in CD4+ T cells from SLE was lower than that from healthy controls (P0.05).The upregulation of miR-126 and its host gene EGFL7 expression in CD4+ T cells from SLE is associated with the hypomethylation of the EGFL7 promoter.

To construct a special luciferase reporter to detect DNA methylation regulatory activity in FCER1G gene promoter regulatory element.We constructed special full and mock methylated FCER1G gene promoter regulatory luciferase reporters by patch-methylation, and detected DNA methylation regulatory activity by comparing the luciferase activity of full-methylated luciferase reporters with mock-methylated reporters.We successfully constructed the full and mock methylated FCER1G gene promoter regulatory luciferase reporters. The ratio of luciferase activity between the full methylated and the mock methylated was (0.36±0.07):1 (P0.001).FCER1G promoter activity is methylation-sensitive and is regulated by DNA methylation.

To observe the demethylation effect of demethylation inhibitor 5-azacytidine (5-Zac) on programmed death receptor 1 (PD-1) in Molt-4 cells (T lymphocyte cell line) and to investigate the relationship between DNA demethylation and expression of PD-1.Molt-4 cells were cultured in the medium containing different concentrations of 5-Zac(0, 5, 10 μmol/L) for 72 h. According to the concentrations of 5-Zac, the Molt-4 cells were divided into a 0 μmol/L 5-Zac group, a 5 μmol/L 5-Zac group, and a 10 μmol/L 5-Zac group. The expression of PD-1 in Molt-4 cells was detected by flow cytometry and the apoptosis rate was calculated. The mRNA transcription level of PD-1 was detected by real-time polymerase chain reaction; Molt-4 cell DNA in all groups were treated by sodium bisulfite. The PD-1 promoter fragment was amplified by PCR, the amplification fragments were transformed into E. coli., the positive clones were selected for equencing, and the methylation status of the fragments of PD-1 promoter was examined. RESULTS Seventy-two hours after the 5-Zac treatment, the expression rate of PD-1 in the Molt-4 cells in the 0 μmol/L 5-Zac group, the 5 μmol/L 5-Zac group, and the 10 μmol/L 5-Zac group was (1.13 ± 0.01)%, (18.96 ± 1.87)%, and (63.09 ± 6.25)% respectively, in a low concentration-dependent way. The PD-1 mRNA expression level was increased significantly with the 5-Zac treatment. Cells apoptosis showed that:compared with the 0 μmol/L 5-Zac group, the apoptosid rate in the 5 μmol/L 5-Zac group and 10 μmol/L 5-Zac group was signficantly increased, which was (1.9 ± 0.06)%, (8.89 ± 1.36)%, and (24.50 ± 3.68)% in the 0 μmol/L 5-Zac group, the 5 μmol/L 5-Zac group, and the 10 μmol/L 5-Zac mol/L group respectively. The bisulfite genomic sequencing showed that the demethylation probability of CpG points on -601 bp and -553 bp was significantly increased in the 5-Zac treated cells compared with those untreated.5-Zac can result in the increase of PD-1 expression in the human lymphoid cell series Molt-4 in vitro, and the apoptosis rate increases, which is related to PD-1 gene promoter demethylation.

Objective To identify microRNA genes with abnormal expression in the CD4+ T cells of patients with systemic lupus erythematosus (SLE) and to determine the role of microRNA-126 (miR-126) in the etiology of SLE. Methods MicroRNA expression patterns in CD4+ T cells from patients with SLE and healthy control subjects were analyzed by microRNA microarray and stem loop quantitative polymerase chain reaction (qPCR). Luciferase reporter gene assays were performed to identify miR-126 targets. Dnmt1, CD11a, and CD70 messenger RNA and protein levels were determined by real-time qPCR, Western blotting, and flow cytometry. CD11a, CD70, and EGFL7 promoter methylation levels were detected by bisulfite sequencing. IgG levels in T cell–B cell cocultures were determined by enzyme-linked immunosorbent assay. Results The expression of 11 microRNA was significantly increased or decreased in CD4+ T cells from patients with SLE relative to that in CD4+ T cells from control subjects. Among these, miR-126 was up-regulated, and its degree of overexpression was inversely correlated with Dnmt1 protein levels. We demonstrated that miR-126 directly inhibits Dnmt1 translation via interaction with its 3′–untranslated region, and that overexpression of miR-126 in CD4+ T cells can significantly reduce Dnmt1 protein levels. The overexpression of miR-126 in CD4+ T cells from healthy donors caused the demethylation and up-regulation of genes encoding CD11a and CD70, thereby causing T cell and B cell hyperactivity. The inhibition of miR-126 in CD4+ T cells from patients with SLE had the opposite effects. Expression of the miR-126 host gene EGFL7 was also up-regulated in CD4+ T cells from patients with SLE, possibly in a hypomethylation-dependent manner. Conclusion Our data suggest that miR-126 regulates DNA methylation in CD4+ T cells and contributes to T cell autoreactivity in SLE by directly targeting Dnmt1.

Interleukin- (IL-)10 and IL-13 play important roles in Th2 cell differentiation and production of autoantibodies in patients with (SLE). However, the mechanisms leading toIL10andIL13overexpression in SLE patients are not well understood. In this study, we confirm that the levels of bothIL10andIL13mRNA in CD4+T cells and of serumIL10andIL13proteins are increased in SLE patients. We show that the DNA methylation levels withinIL10andIL13gene regulatory domains are reduced in SLE CD4+T cells relative to healthy controls and negatively correlate withIL10andIL13mRNA expression. Moreover, treating healthy CD4+T cells with the demethylating agent 5-azacytidine (5-azaC) increasedIL10andIL13mRNA transcription. Together, our results show that promoter methylation is a determinant ofIL10andIL13expression in CD4+T cells, and we propose that DNA hypomethylation leads toIL10andIL13overexpression in SLE patients.