Direct measurement of B-cell receptor repertoire's composition and variation in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that is known to be associated with polyclonal B-cell hyper-reactivity. B-cell receptor (BCR) has a central role in B-cell development, activation, survival and apoptosis, and thus is a critical component of the regulation of both protective and autoreactive B cells. In this study, we applied multiplex PCR and Illumina high-throughput sequencing to study the composition and variation of the BCRs in peripheral blood mononuclear cells from SLE patients and healthy donors (NC). We found that SLE group displayed significantly shorter CDR3 average length (14.86±0.76aa vs 15.70±0.43aa), more arginine percentage of CDR3 amino acids (7.57±0.20% vs 7.32±0.19%) and poorer immunological diversity than the healthy ones. CDR3 sequence YGMDV present in all SLE samples may provide more information in generating more effective B-cell targeted diagnosis/therapies strategies.

[Carpal canal ultrasound examination in patients with mild hand-arm vibration disease].

Objective: To investigate the clinical value of ultrasound examination of carpal canal structure in patients with mild hand-arm vibration disease. Methods: A total of 29 patients (58 wrists) with mild hand-arm vibration disease who were treated in Shenzhen Prevention and Treatment Center for Occupational Diseases from May to December, 2015 were enrolled as observation group, and 20 healthy volunteers (40 wrists) were enrolled as the control group. Color Doppler ultrasound was used to observe the morphology and echo of the median nerve in the carpal canal and 9 muscle tendons and transverse carpal ligament. The thickness of transverse carpal ligament and diameter of the median nerve at the level of the hamulus of hamate bone were measured, as well as the cross-sectional area of the median nerve at the level of pisiform bone. Results: In the 29 patients with hand-arm vibration disease patients in the observation group, 8 experienced entrapment of the median nerve in the carpal canal, among whom 5 had entrapment in both wrists; there were 13 wrists (23%) with nerve entrapment and 45 wrists (77%) without nerve entrapment. Compared with the control group, the patients with hand-arm vibration disease and nerve entrapment in the observation group showed significant thickening of the transverse carpal ligament at the level of the hamulus of hamate bone and a significant increase in the cross-sectional area of the median nerve at the level of pisiform bone (P<0.05) , while there were no significant differences in the thickness of transverse carpal ligament at the level of the hamulus of hamate bone and the cross-sectional area of the median nerve at the level of pisiform bone (t=-9.397 and -4.385, both P>0.05) . Conclusion: Ultrasound examination can clearly show the radiological changes of carpal canal contents in patients with mild hand-arm vibration disease and has a certain diagnostic value in nerve damage in patients with hand-arm vibration disease.

A multicentre, randomized, single-blind comparison of topical clindamycin 1%/benzoyl peroxide 5% once-daily gel versus clindamycin 1% twice-daily gel in the treatment of mild to moderate acne vulgaris in Chinese patients.

BACKGROUND: Acne vulgaris affects up to 54% of Chinese adolescents. Combination therapy has become the recommended standard of care for acne. OBJECTIVE: The aim of this study was to compare the efficacy and safety of clindamycin (1%) and benzoyl peroxide (5%) (CDP/BPO) gel once daily vs. clindamycin (1%) (CDP) monotherapy gel twice daily in Chinese patients with mild to moderate acne. METHODS: 1020 patients (aged 12-45 years) with mild to moderate acne were randomized (1 : 1); 1016 patients were treated with CDP/BPO (n = 500) or CDP (n = 516) for a 12-week treatment period. Efficacy assessments were performed at baseline, and at weeks 1, 2, 4, 8 and 12; and primarily included change in total lesion count (inflammatory and non-inflammatory lesions), and proportion of patients with a minimum 2-grade improvement in Investigator's Static Global Assessment (ISGA) score. Patient safety and local tolerability were also evaluated. RESULTS: Patients in CDP/BPO group showed a greater per cent reduction in total lesion count compared with patients in CDP group at week 12 (delta = -0.05; 95% CI = -0.09, -0.02; P = 0.003); statistically significant reduction in lesion count was noted as early as week 1 and continued through week 12. A greater proportion of patients in CDP/BPO group showed a ≥2-grade improvement in ISGA score at week 12 compared with CDP group (30.2% vs. 22.7%; P = 0.018). Overall, the incidence of adverse events (AEs) was higher in the CDP/BPO group (14.4%) than in the CDP group (7.9%); the most commonly reported events were generally related to application site reactions (erythema, pruritus and swelling). Incidence of drug-related AEs was 8.6% in CDP/BPO group and 1.2% in CDP group. Both groups showed trends towards reduction in investigator and subject rated local tolerability scores. CONCLUSION: CDP/BPO gel demonstrated superior efficacy over CDP gel along with acceptable safety and tolerability in Chinese patients with mild to moderate acne. GOV NUMBER: NCT01915732.

DNA hypermethylation of the forkhead box protein 3 (FOXP3) promoter in CD4+ T cells of patients with systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a complex autoimmune disease that involves dysregulation of immune homeostasis. The failure of impaired regulatory T cells (Tregs) to maintain immune homeostasis plays a major role in the development of SSc. Transcriptional silencing of the forkhead box protein 3 gene (FOXP3) via hypermethylation of regulatory regions has been identified as a hallmark of committed Tregs and several autoimmune disorders. OBJECTIVES: To investigate whether aberrant expression and methylation of FOXP3 occurs in CD4+ T cells of patients with SSc and their roles in the pathogenesis of SSc. METHODS: FOXP3 expression in CD4+ T cells was measured by real-time quantitative reverse-itranscriptase polymerase chain reaction and western blot. Bisulfite sequencing was performed to determine the methylation status of the FOXP3 proximal promoter sequence. The percentage of Treg cells was estimated by flow cytometry. RESULTS: Decreased FOXP3 expression was observed in CD4+ T cells from patients with SSc. The methylation levels of the FOXP3 regulatory sequences were elevated and inversely correlated with FOXP3 mRNA expression in patients with SSc. The number of Tregs was significantly reduced in patients with SSc. Treatment of SSc CD4+ T cells with a DNA methylation inhibitor, 5-azacytidine, reduced the mean methylation levels, and enhanced FOXP3 expression and Treg generation. The promoter methylation status and expression level of FOXP3 are significantly associated with disease activity. CONCLUSIONS: The contribution of the hypermethylation of the FOXP3 promoter to decreased FOXP3 expression and the subsequent quantitative defects of Tregs may mediate the immune dysfunction in SSc.

Down-regulation of mitochondrial ATPase by hypermethylation mechanism in chronic myeloid leukemia is associated with multidrug resistance.

BACKGROUND: To identify novel proteins involved in multidrug resistance in chronic myeloid leukemia (CML). MATERIALS AND METHODS: Comparative proteomics was used to screen multidrug resistance-related proteins from K562 and K562/A02; the differently expressed proteins were further confirmed by western blot and real-time PCR. short hairpin RNA (shRNA) assay was applied to determine the relationship between candidate protein and adriamycin resistance. Bisulfite sequencing was carried out to assess methylation status of candidate multidrug resistance-related gene promoter. K562/A02 was treated with 5-azacytidine or trichostatin A (TSA); multidrug resistance phenotype and corresponding protein or gene changes were detected. RESULTS: Seventeen proteins with altered abundances of more than twofold were detected, among which mitochondrial ATPase in K562/A02 was significantly down-regulated. Suppressing mitochondrial ATPase by shRNA could enhance adriamycin resistance and antiapoptosis activity of K562. The promoter hypermethylation in mitochondrial ATPase was found to be attributed to the adriamycin-resistant phenotype of both K562/A02 (methylated frequency 18.18%) and CML primary cells in accelerated phase (methylated frequency 7.95%) or blast crisis (methylated frequency 26.59%). Inhibition of hypermethylation increased adriamycin sensitivity of K562/A02. A synergistic effect on reversing adriamycin-resistant phenotype was obtained when 5-azacytidine was combined with TSA. CONCLUSION: Down-regulation of mitochondrial ATPase can lead to adriamycin resistance in CML and the mechanism is associated with DNA methylation regulation.

The effects of glaucoma filtering surgery on anterior chamber-associated immune deviation and contribution of lymphatic drainage in rats.

PURPOSE: To investigate whether glaucoma filtering surgery (GFS) in rats would impair the eye's capacity to induce anterior chamber-associated immune deviation (ACAID) and assess the possible mechanism involved. METHODS: Rats subjected to GFS were injected with bovine serum antigen (BSA) into the anterior chamber to induce ACAID. Animals that had their cervical lymph nodes (CLNs) excised before filtering surgery and those that had sham filtering surgery served as control comparison groups. Antigen-specific delayed-type hypersensitivity (DTH) was used to identify the induction of ACAID. Antigen level in the CLNs was indicated by the percentage of FITC-positive cells in CLNs after FITC dextran was injected into the anterior chamber. Statistical analyses were performed using the student's t-test for comparison of data between control and experimental groups. A P<0.05 was required for results to be considered statistically significant. RESULTS: Rats undergoing GFS demonstrated antigen-specific DTH, while those in the sham filtering surgery or CLNs excised groups failed to acquire an antigen-specific DTH response. The percentage of FITC-positive cells in CLNs was significantly increased (P=0.001) in GFS (mean+/-SD: 2.96+/-0.67%) vs controls (1.57+/-0.48%) at 1 day, but not at 3, 5, 7, or 12 days post-antigen injection. CONCLUSIONS: GFS prevents the induction of ACAID in rats, and the antigen drainage to CLNs plays a critical role in this process. The results suggest that the ocular immune status might be altered by GFS.

[Construction of recombinant antisense RNA IgG2aVH region of murine systemic lupus erythematous].

OBJECTIVE: To construct recombinant plasmid of IgG2aVH region antisense RNA of systemic lupus erythematous. METHODS: Total RNA was isolated from spleen cell of BWF1 mice. Using spleen cell total RNA as the template, We designed specific primers from A6.1 region sequences, and amplified the IgG2aVH region 375 bp DNA fragments by RT-PCR. The IgG2aVH cDNA was cloned by T/A and inserted into pcDNA 3.1 plasmid of vector. RESULTS: The IgG2aVH antisense RNA plasmid expressing recombinant was identified by restriction enzyme digestion and DNA sequence analysis. CONCLUSION: There is IgG2aVH gene in F1 mice spleen cell; the IgG2aVH antisense RNA expressing recombinants is constructed successfully.

[Establishment and its application of a reporter-based screening model for discovering new ligands of estrogen receptor alpha subtypes].

AIM: To establish a sensitive and efficient reporter gene based screening model and use it to screen compounds for discovering new ligands of estrogen receptor alpha subtype. METHODS: A recombinant Epstein-Barr virus episomal vector (pMT/ERE-CAT) was constructed by inserting a synthetic sequence composed of five estrogen response elements upstream of promoter and a chloramphenicol acetyltransferase (CAT) gene downstream of promoter. pMT/ERE-CAT was transfected into HepG2 cells expressing estrogen receptor alpha subtype (ER +HepG2). Hygromycin (200 micrograms.mL-1) was added 48 h after transfection for selection. One stably transfected clone was isolated and used to screen compounds for activity of stimulating CAT gene expression using colorimetric CAT assay. RESULTS: In the ER +HepG2 cells, the expression of CAT gene was induced by estradiol. A dose-dependent expression of CAT gene with half-maximal induction at 0.07 nmol.L-1 was observed. The ER +HepG2 cell was used to screen compounds for activity of stimulating CAT gene expression. Resveratrol was found to produce a maximal level of induction (1.75 times of estradiol). In vitro radiation survival experiment showed that the radioprotection activity of resveratrol (D0 = 3.18 Gy) is stronger than that of estradiol (D0 = 2.59 Gy). CONCLUSION: Vector pMT/ERE-CAT was used to generate stably transfected ER +HepG2 cell lines. The cell lines can be used to screen compounds for estrogen activity by testing extracts of cells grown in microtiter wells directly using colorimetric CAT assay. This system should provide an efficient method for screening and analyzing the activity of large numbers of ligands of estrogen receptor.