Objective:To observe the expression of BTLA on T cells during activation and further analyze its inhibitory effects on T cell activation in different phases.Methods: T cells from PBMC were enriched by negative selection using magnetic beads.Expression of BTLA,CTLA-4 and PD-1 on freshly isolated human T cells and kinetics expression of BTLA,CILA-4 and PD-1 on CD3 mAb stimulated T cells were examined by flow cytometry.T cells were stimulated by anti-CD3 mAb combined with anti-CD28 mAb in the presence of anti-BTLA mAb 8H9,then T cell proliferation was tested by MTT assay in the different culture time.Immature DCs were generated from monocytes cultured in the medium containing GM-CSF and IL-4, and further driven to maturation by anti-CD40 mAb.Expression of HVEM on DCs was measured by flow cytometry.T cells were co-cultured with DCs in the presence of soluble 8H9 or anti-HVEM antibody to block HVEM-BTLA interaction,T cell proliferation was measured by MTT assay.Results:Freshly isolated T cells exhibited high levels of BTLA expression, but not CTLA-4 and PD-1.After T cell activation, BTLA expression decreased on first 2 days, with rapidly increasing to high levels.Unlike BTLA, expression of CTLA-4 and PD-1 was gradually increased during T cell activation.8H9 significantly inhibited the proliferation of T cell stimulated by CD3 mAb and CD28 mAb.8H9 could still exhibit inhibitory effect on T cell proliferation after 24 h or 48 h of preactivation by CD3 mAb plus CD28 mAb stimulation.HVEM was highly expressed on immature DCs, and down-regulated on mature DCs.Blockade of BTLA by soluble 8H9 or anti-HVEM antibody enhanced DC-mediated T cell proliferation within 48 h.Conclusion: BTLA signal enhances the threshold of T cell activation and plays importantly negative regulatory role in the initiation and early phase of T cell activation.

Objective To investigate the expression of Signal transducer and activator of transcription 2 (STAT2) gene in the peripheral blood mononuclear cells of patients with systemic lupus erythematosus, and to evaluate the possible connections between STAT2 gene expression levels and clinical features. Methods One hundred and forty-four SLE patients, 27 non-SLE patients with other rheumatisms and 58 normal controls were recruited for this research, and the subjects were surveyed for clinical data collection. SYBR Green Dye based real-time quantitative PCR method was used to compare the expression levels of STAT2 in patients with SLE and those in the controls. The correlation of the gene expression levels and disease activity and specificity was studied. Results STAT2 expression levels (5.2±1.7) in SLE patients were remarkably higher than those in non-SLE patients and normal controls (4.3±1.1, 4.5±1.2, P<0.01 in both). The expression levels of STAT1 were increased in active SLE patients(5.2±1.5), comparing with those observed in inactive SLE patients (4.8±2.9, P<0.01), and expression levels of STAT1 in SLE patients were negatively correlated with C3 levels in sera (r=-0.449, P<0.01) whereas were positively correlated with SLEDAI-2K score and 24 hour urine protein (r=0.317, 0.309, P<0.01 in both). Conclusion Over-expression of STAT2 gene in the peripheral blood cells is linked with the pathogenesis of systemic lupus erythematosus, and the elevated expression level of STAT2 is correlated with SEE disease activity.

Objective: To prepare anti-VSIG4 monoclonal antibodies and characterize their biological functions.Methods: BALB/c mice were immunized with transfected cell line (L929/VSIG4L) as immunogen.The spleen B cells of the mice were fused with SP2/0 and hybridoma cells were screened with transfected cell line (L929/VSIG4) by FCM.After acquisition of the hybridomas secreting anti-VSIG4 mAb,their biological activities were investigated by indirect immunofluorescence,Western blot,competitive inhibition test,and MTT assay.Results:Two stable hybridomas,9A7 and 9D5 were obtained,which could continuously secrete specific anti-VSIG4 monoclonal antibodies.The following biological activity studies showed that these monoclonal antibodies could recognize the natural VSIG4 expressed on the macrophages and several cancer cell lines,such as Jurkat,THP-1 and H446.Furthermore,they could block the inhibitory effects of VSIG4 on proliferation of T cells in vitro.Conclusion: Two hybridomas secreting anti-VSIG4 monoclonal antibodies have been established.These monoclonal antibodies provide useful tools for further studying VSIG4's biological function and its unknown receptor.

Objective To correlate the expression levels of interferon inducible genes (IFIGs) with disease activity and clinical features in systemic lupus erythematosus (SLE) patiems,.Methods Peripheral blood cells obtained from 67 SLE patients and 23 healthy donors (HDs) were subjected to real-time PCR to measure the transcriptional levels of five IFIGs (OAS-1,Mx-1,Ly6e,IFIT1 and IFIT4).Interferon scores were calculated and were compared between various groups of SLE patients as well as between patients and controls;ISRE lucife:rase reporter gene activity was measured in 17 of 67 patients and correlated with interferon score.Results Interferon scores were strongly correlated with ISRIE reporter gene aetivity,which represented for the type Ⅰ interferon activity in serum.The expression.levels of IFIGs and jinterferon scores were significantly elevated in SLE patients compared with HDs (P＜0.0001).Interferon scores were correlated positively with SLEDAI-2K(P=0.0006) and negatively with C3 levels(P=0.0162).Interferon scores were also significantly elevated in SLE patients with a positive anti-Sm or anti-RNP autoantibodies.Clonclusion The interferon score may be regarded as a good indicator for serum type I interferon activity in SLE and serves as a new hiomarker for disease activity in SLE patients.

ObjectiveTo further understand the clinic manifestations of childhood primary Sjogren's Syndrome(pSS) and enhance early diagnosis. MethodsFive cases of pSS from Renji Hospital, Shanghai, were reported and their clinical features were analysoed. And literatures from Medline database and Weipu database were reviewed and discussed. Results①Childhood pSS had various clinic presentations that were non-specific and sicca symptoms were absent or occur late in most cases. ② The most common presentations were recurrent parotiditis and cutaneous manifestations with various locations and forms. ③ American-European Criteria for SS were not suitable for the diagnosis of childhood pSS. ConclusionRecurrent parotiditis and cutaneous manifestations in children can be used as clues for the diagnosis of childhood pSS but needs to be further confirmed by the positive results of salivary gland biopsy and autoantibodies examination, particularly SSA/SSB.

Objective To explore the effects of systemic lupus erythematosus (SLE) susceptibility gene IFIT1 on chemokine expression in RAW264.7 macrophages and its possible role in the pathogenesis of SLE. Methods The expression vector of pEGFP-N1/IFIT1 was transfected into RAW264.7 cells by electroporation. 24 h after transfection, cells were stimulated with LPS ( 1 μg/ml). The transcriptional levels of chemokine MIP-1α, RANTES, CCL9, CXCL2 and IP-10 were measured at various time points after stimu-lation using real-time quantitative PCR. The chemokine expression levels in the kidneys of 8 week-old NZB/NZW F1 mice were also determined by real time PCR. Results Compared with cells transfected with null vector, IFIT1 high RAW264.7 cells produced significantly increased levels of MIP-1α, RANTES, CCL9, CXCL2 and IP-10 both at 4 h and 24 h after stimulation (P<0.05). Chemokine expression levels were signi-ficantly elevated in kidneys of 8 week-old NZB/NZW F1 mice compared with those of 8 week-old BALB/c mice controls (P<0.05). Conclusion IFIT1 may participate in target organ damages in SLE via augmentation of chemokine production by macrophage cells.

Objective To explore the pathogenic genes relevant to Behcet's disease (BD) by building the differentail gene expression profiles of peripheral blood leukocytes in BD. Methods Oligonucleotide gene array from Affymetrix Company was applied to study the differed expression levels of whole genome between three age and sex matched BD patients and normal controls. Four genes, BCL6, LRAP, ICOSLG and MME, were selected to be tested for gene expression levels by real-time PCR in the groups of BD, normol controls (NC), Lupus and rheumatoid arthritis (RA) peticnts. Results ① Differential gene expression profile of BD compared to that of normal controls was built up. It contained 89 up-regulated and 57 down-regulated genes. ② Four genes mentioned above had significantly higher expression levels in active BD patients than those in NC but had lower exoression levels in stable BD patients. The expression levels of BCL6 and MME were also proved to be increased significantly in BD than in RA and SLE patients. Conclusion ① Our work shed some light on further research of the etiopathogenesis of BD. ② The expression levels of the four genes are proved to be relevant to BD the first time by us. Further analysis showes that TNF-α and IFN-γ can up-regulate the expression levels of BCL6, LRAP and ICOSLG which may be novel to BD. The MME gene is expressed on the surface of cells, which is convenient for test and may potentially be a marker for the diagnosis of BD.

Objective: To develop a molecular screening test for genetic defects on hearing loss related genes has significant impacts on early identification of hereditary hearing loss and genetic susceptibility to aminoglycoside ototoxicity. Early identification of pre-lingual hearing loss is very important for patient's language development, academic achievement, and social skill. Two common mutations, the 235delC in GJB2 gene and the mutation A1555G in mitochondrial DNA, are included in the newly developed screening panel for Chinese population. Methods: A molecular genetic assay, based on fluorescent labeled multiplex PCR and automatic DNA fragment analyzing techniques, was developed to detect both mutations simultaneously. Results: This assay was able to detect both mutations from patient's samples, and pooled DNA tests, as well as suitable to detect mutation from the DNA extracted from dried blood spot and buccal swab. Conclusion: This assay could be a useful tool for newborn screening and carrier screening for the hereditary hearing loss for the Chinese population.

OBJECTIVETo develop an improved substrate for indirect immunofluorescence test (IIF) for detecting anti-Ro60/Sjogren's syndrome A (Ro/SSA) autoantibodies.

METHODS60-kDa Ro/SSA autoantigens (Ro60) cDNAs were obtained from human placental cDNA library using polymerase chain reaction (PCR) and were cloned into the mammalian expression vector-pEGFP-C1. Then, the recombinant plasmids were transfected into HEp-2 cells. We confirmed the overexpression, localization and antigenicity of fusion proteins in transfected cells by means of immunoblotting, confocal fluorescence microscopy and IIF. HEp-2 and HEp-Ro60 were analyzed by IIF using a panel of 10 precipitin-positive anti-Ro human sera simultaneously.

RESULTSStable expression of Ro60-green fluorescent protein (Ro60-GFP) fusion proteins were maintained ten more generations. Ro60-GFP kept the antigenicity of Ro while demonstrating its own characteristic immunofluorescent pattern in HEp-Ro60 cells. The transfectants dramatically increased the sensitivity of IIF testing (a mean increase of 6.7-fold in endpoint titer). Eight over ten (8/10) positive anti-Ro sera showed characteristic immunofluorescent patterns for HEp-Ro60, including two sera that were anti-nuclear antibodies (ANA) negative for untransfected HEp-2. IIF-ANA in all healthy sera was negative for HEp-Ro60.

CONCLUSIONSAs a new substrate for IIF, the Ro60 transfectants can be used to detect anti-Ro antibodies. In addition, transfected HEp-2 cells keep the immunofluorescent properties of HEp-2 cells in IIF-ANA tests and can be employed as a substrate for routine IIF-ANA detection.

Antibodies, Antinuclear ; blood ; Autoantigens ; Cell Line ; Fluorescent Antibody Technique, Indirect ; Humans ; Molecular Weight ; RNA, Small Cytoplasmic ; Recombinant Fusion Proteins ; immunology ; Ribonucleoproteins ; immunology ; Transfection

OBJECTIVETo evaluate the efficacy and safety of leflunomide in comparison with methotrexate (MTX) on patients with rheumatoid arthritis (RA) in China.

METHODSFive hundred and sixty-six patients with active rheumatoid arthritis were randomly assigned to receive leflunomide at 20 mg once daily or MTX at 15 mg once weekly in a controlled trial. Five hundred and four patients completed the 12-week treatment and some patients continued the treatment for 24 weeks.

RESULTSBoth leflunomide and MTX could improve the symptoms, signs, and joint function, but there were no changes in X-ray observations of patients with rheumatoid arthritis. In the leflunomide group, the overall rates of effectiveness at 12 weeks and 24 weeks were 86.94% and 92.31% respectively; the rates of remarkable improvement were 64.95% and 79.81% respectively. In the MTX group, the overall rates of effectiveness at 12 weeks and 24 weeks were 84.04% and 83.15% respectively; the rates of remarkable improvement were 56.81% and 75.28% respectively. According to intent-to-treat analysis, the ACR 20% response rates at 12 weeks and 24 weeks in the leflunomide group were 62.54% and 67.18% respectively, compared with 60.08% and 61.32% respectively in MTX group. No statistical differences were shown in the efficacy between the two groups (P > 0.05). The adverse events in the leflunomide group were gastrointestinal symptoms, skin rash, alopecia, nervous system symptoms, decreased leukocyte count, and elevation of alanine aminotransferase (ALT). Most of these side effects were mild and transient. The incidence of adverse events in the leflunomide group was 16.84%, significantly lower than that in MTX group (28.17%, P = 0.002).

CONCLUSIONSLeflunomide is effective in the treatment of RA with less adverse events than MTX. Its efficacy is similar to MTX, but the incidence of adverse events and the rate of withdrawal due to adverse events were lower in the leflunomide group than in MTX group.

Antirheumatic Agents ; adverse effects ; therapeutic use ; Arthritis, Rheumatoid ; drug therapy ; Female ; Growth Inhibitors ; adverse effects ; therapeutic use ; Humans ; Immunosuppressive Agents ; adverse effects ; therapeutic use ; Isoxazoles ; adverse effects ; therapeutic use ; Male ; Methotrexate ; adverse effects ; therapeutic use ; Middle Aged