Ming Fei Liu

Decreased CD4+CD25+ T cells in peripheral blood of patients with systemic lupus erythematosus.

Recent animal studies have shown that CD4+CD25+ T cells play a crucial role in the suppression of the immune response and that depletion of this subset of T cells might lead to development of autoimmune diseases. The aim of the present study was to investigate the levels of CD4+CD25+ T cells in the peripheral blood of patients with systemic lupus erythematosus (SLE). Ninety‐four SLE patients, 52 patients with rheumatoid arthritis (RA) and 50 age‐ and gender‐matched healthy individuals were enrolled in the study. A flowcytometric method was applied in the measurement of CD4+CD25+ T cells. The results showed that patients with SLE had statistically lower levels of CD4+CD25+ T cells than did normal controls, when expressed as either percentages of peripheral blood mononuclear cells (PBMCs) (mean ± SD, 8.49 ± 6.36 versus 11.11 ± 4.58%, P < 0.05) or absolute cell numbers (98.77 ± 97.52 versus 213.93 ± 104.52 cells/mm3, P < 0.05). In terms of CD25brightCD4+ T cells, defined as having a fluorescence intensity of CD25 expression exceeding 100, SLE patients still had significantly lower levels than did normal controls expressed as percentages of PBMCs (1.76 ± 1.32 versus 3.73 ± 1.30%, P < 0.05). No significant differences could be found between RA patients and normal controls. The overwhelming majority of CD4+CD25+ T cells belonged to CD45RO+ cells and most did not express the CD69 molecule. Although decreased CD4+CD25+ T cells were found in SLE patients, we failed to find a significant correlation between the levels of CD4+CD25+ T cells and disease activities of SLE. To the best of our knowledge, this is the first study to demonstrate that patients with SLE had decreased CD4+CD25+ T cells. However, the exact role of the decreased CD4+CD25+ T cells in the pathogenesis of SLE remains to be elucidated.

Increased expression of soluble cytotoxic T-lymphocyte-associated antigen-4 molecule in patients with systemic lupus erythematosus.

A soluble form of cytotoxic T‐lymphocyte‐associated antigen‐4 (sCTLA‐4) was recently found and shown to possess a downregulatory function as a membrane‐bound CTLA‐4 molecule. The purpose of the study was to investigate the expression of sCTLA‐4 molecule in patients with systemic lupus erythematosus (SLE). One hundred patients with SLE and 40 age‐ and sex‐matched healthy individuals were enrolled in the study. The results showed that patients with SLE have significantly higher levels of sCTLA‐4 in sera than healthy controls (21.6 ± 12.3 ng/ml versus 5.9 ± 5.4 ng/ml, P < 0.001). Increased expression of sCTLA‐4 mRNA in peripheral blood mononuclear cells (PBMCs) was also found in SLE patients. However, we could not find a statistically significant correlation between the serum levels of sCTLA‐4 and lupus disease activities. The reported CTLA‐4 gene polymorphism in promoter region at position −318 did not affect the levels of sCTLA‐4. To the best of our knowledge, this is the first report showing that patients with SLE have increased sCTLA‐4 expression. However, the mechanism and role of increased sCTLA‐4 in the pathogenesis of SLE remains elucidated.

Brief Report: Amelioration of collagen-induced arthritis in mice by lentivirus-mediated silencing of microRNA-223

OBJECTIVE MicroRNA (miRNA) plays a role in autoimmune diseases. MiRNA-223 (miR-223) is up-regulated in patients with rheumatoid arthritis (RA) and is involved in osteoclastogenesis, which contributes to erosive disease. The aim of this study was to test the feasibility of using lentiviral vectors expressing the miR-223 target sequence (miR-223T) to suppress miR-223 activity as a therapeutic strategy in a mouse model of collagen-induced arthritis (CIA). METHODS Levels of miR-223 in the synovial tissue of patients with RA or osteoarthritis (OA), as well as in the ankle joints of mice with CIA, were determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Lentiviral vectors expressing miR-223T (LVmiR-223T) or luciferase short hairpin RNA (LVshLuc) as a control vector were injected intraperitoneally into mice with CIA. Treatment responses and disease-related bone mineral density were monitored. Levels of nuclear factor 1A (NF-1A), a direct target of miR-223, and macrophage colony-stimulating factor receptor (M-CSFR), which is critical for osteoclastogenesis, were measured by immunohistochemistry and quantitative RT-PCR. Osteoclasts were assessed by tartrate-resistant acid phosphatase staining. RESULTS MiR-223 expression was significantly higher in the synovium of RA patients and in the ankle joints of mice with CIA as compared to OA patients and normal mice. LVmiR-223T treatment reduced the arthritis score, histologic score, miR-223 expression, osteoclastogenesis, and bone erosion in mice with CIA. Down-regulation of miR-223 with concomitant increases in NF-1A levels and decreases in M-CSFR levels was detected in the synovium of LVmiR-223T-treated mice. CONCLUSION This study is the first to demonstrate that lentivirus-mediated silencing of miR-223 can reduce disease severity of experimental arthritis. Furthermore, our results indicate that inhibition of miR-223 activity should be further explored as a therapeutic strategy in RA.

The Presence of Cytokine‐Suppressive CD4+CD25+ T Cells in the Peripheral Blood and Synovial Fluid of Patients with Rheumatoid Arthritis

CD4+CD25+ T cells have been shown to play a regulatory or suppressive role in the immune response and are possibly relevant to the pathogenesis of autoimmune diseases. In the present study, we attempted to investigate the levels of CD4+CD25+ T cells in the peripheral blood (PB) and synovial fluid (SF) of patients with rheumatoid arthritis (RA) and the effects of CD4+CD25+ T cells on the in vitro cytokine production by stimulated SF mononuclear cells (SFMC). The results showed that RA patients had similar frequencies of CD4+CD25+ T cells in PB, expressed as a percentages of the lymphocyte population, as did healthy subjects (mean ± SD: 10.52 ± 5.87% versus 11.11 ± 4.58%., respectively). But in contrast to PB, the SF of RA patients contained significantly higher levels of CD4+CD25+ T cells (17.77 ± 7.92% versus 10.52 ± 5.87%, respectively. P < 0.001). When cocultured in vitro with SFMC, CD4+CD25+ T cells purified from either PB or SF were found to exert a considerable suppressive effect on the production of cytokines including TNF‐α, IFN‐γ and interleukin‐10 (IL‐10). The percentages of inhibition of each cytokines ranged from 41.8 to 98.4% (mean, 80.0%) for TNF‐α, 42.8 to 98.9% (mean, 83.2%) for IFN‐γ and 59.3 to 96.6% (mean, 80.0%) for IL‐10. Because both pro‐inflammatory and anti‐inflammatory cytokines were suppressed by CD4+CD25+ T cells, whether CD4+CD25+ T cells might play a beneficial role in the suppression of sustained inflammation in rheumatoid synovium remains to be elucidated.

Use of HLA-B*58:01 genotyping to prevent allopurinol induced severe cutaneous adverse reactions in Taiwan: national prospective cohort study

Objective To evaluate the use of prospective screening for the HLA-B*58:01 allele to identify Taiwanese individuals at risk of severe cutaneous adverse reactions (SCARs) induced by allopurinol treatment. Design National prospective cohort study. Setting 15 medical centres in different regions of Taiwan, from July 2009 to August 2014. Participants 2926 people who had an indication for allopurinol treatment but had not taken allopurinol previously. Participants were excluded if they had undergone a bone marrow transplant, were not of Han Chinese descent, and had a history of allopurinol induced hypersensitivity. DNA purified from 2910 participants’ peripheral blood was used to assess the presence of HLA-B*58:01. Main outcome measures Incidence of allopurinol induced SCARs with and without screening. Results Participants who tested positive for HLA-B*58:01 (19.6%, n=571) were advised to avoid allopurinol, and were referred to an alternate drug treatment or advised to continue with their prestudy treatment. Participants who tested negative (80.4%, n=2339) were given allopurinol. Participants were interviewed once a week for two months to monitor symptoms. The historical incidence of allopurinol induced SCARs, estimated by the National Health Insurance research database of Taiwan, was used for comparison. Mild, transient rash without blisters developed in 97 (3%) participants during follow-up. None of the participants was admitted to hospital owing to adverse drug reactions. SCARs did not develop in any of the participants receiving allopurinol who screened negative for HLA-B*58:01. By contrast, seven cases of SCARs were expected, based on the estimated historical incidence of allopurinol induced SCARs nationwide (0.30% per year, 95% confidence interval 0.28% to 0.31%; P=0.0026; two side one sample binomial test). Conclusions Prospective screening of the HLA-B*58:01 allele, coupled with an alternative drug treatment for carriers, significantly decreased the incidence of allopurinol induced SCARs in Taiwanese medical centres.

Measurement of synovial tumor necrosis factor-alpha in diagnosing emergency patients with bacterial arthritis

Because of the high morbidity and mortality in patients with bacterial arthritis, rapidly and correctly diagnosing this critical condition is a challenge to emergency clinicians. Synovial fluid samples were obtained from 75 patients with arthritis disorders who presented to an emergency service, and levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6) were measured. Twenty patients with culture-proven bacterial arthritis had higher levels of synovial TNF-alpha than patients with osteoarthritis or with inflammatory arthritis, including gouty arthritis, rheumatoid arthritis, reactive arthritis, and lupus arthritis. There was a good sensitivity for synovial TNF-alpha level in diagnosing patients with bacterial arthritis. Nearly 100% of patients with bacterial arthritis had elevated synovial TNF-alpha levels. However, synovial IL-1 beta and IL-6 levels failed to discriminate bacterial arthritis from other inflammatory arthritis. Measurement of synovial TNF-alpha level may be useful as a diagnostic aid in emergency patients with bacterial arthritis disorders.

Expression of CD40 and CD40 Ligand among Cell Populations within Rheumatoid Synovial Compartment

Augmented and prolonged expression of CD40 ligand (CD40L) was recently reported in lymphoid cells from human lupus patients, suggesting that CD40/CD40L pathway was involved in the pathogenesis of systemic autoimmune diseases. This study was thus designed to study the expression of CD40 and CD40L among cell populations within inflammatory joints of patients with rheumatoid arthritis (RA). The result showed that most B cells and monocytes in synovial fluids (SF) expressed CD40. Cultured synovial fibroblasts also stained positive for CD40. Regarding CD40L, we found that T cells as well as B cells could express CD40L. Compared with normal controls, RA patients had higher levels of CD40L+ T cells (8.71 ± 17.69 % vs 1.74 ± 2.30 %, P± 0.05) and CD40L+ B cells (7.71 ± 7.64 % vs 1.12 ± 1.59 % P < 0.05). After in vitro stimulation, T cells from RA patients had higher and longer CD40L expression than T cells from normal peripheral blood. For investigating the effect of CD40 expressed on synovial fibroblasts on TNF-a production in joint compartment, we used anti-CD40 antibody to bind CD40 on fibroblasts for one hour and then co-cultured with synovial fluid mononuclear cells. We found that the levels of TNF-a decreased in the presence of anti-CD40 antibody. We concluded that there was an intrinsic hyperexpression of CD40L on lymphoid cells within rheumatoid joints, and synovial fibroblasts could contribute to articular inflammation through surface CD40 molecule.

Incidence of cancer in a nationwide population cohort of 7852 patients with primary Sjogren's syndrome in Taiwan

Objectives Patients with primary Sjögrens syndrome (pSS) are at a higher risk of developing non-Hodgkins lymphoma (NHL). However, little is known with regard to the risk of developing cancers other than NHL. The authors aimed in this study to compare the incidence of cancer in various sites among patients with pSS with the general population of Taiwan. Methods The authors used National Health Insurance claims data to establish a nationwide population cohort of 7852 patients with pSS from 2000 to 2008 who did not have cancer prior to diagnosis of pSS. Incidence and standardised incidence ratios (SIRs) for cancer in various sites were calculated. Results Among patients with pSS, 277 (2.9%) developed cancer. The SIR for cancer was 1.04 (95% CI 0.91 to 1.18) among patients of all ages with pSS and was 2.19 (95% CI 1.43 to 3.21) for patients aged 25–44 years. Female patients with pSS had a higher risk of NHL (SIR 7.1, 95% CI 4.3 to 10.3), multiple myeloma (SIR 6.1, 95% CI 2.0 to 14.2) and thyroid gland cancer (SIR 2.6, 95% CI 1.4 to 4.3) and a lower risk of colon cancer (SIR 0.22, 95% CI 0.05 to 0.65). In contrast, male patients with pSS were not at a higher risk of developing cancer in particular sites. Conclusion Patients with pSS, overall, did not have higher risk of cancer, and only patients aged 25–44 years were at an increased risk of cancer compared with their counterparts in the general population. Cancer screening for patients with pSS, especially female patients, should focus on NHL and multiple myeloma and thyroid gland cancer.

CTLA-4 gene polymorphism in promoter and exon-1 regions in Chinese patients with systemic lupus erythematosus.

Cytotoxic T lymphocyte associated antigen 4 (CTLA-4), a structural homologue of CD28, has been reported to be an important negative regulator of autoimmune diseases. Recent studies showed that CTLA-4 gene polymorphism was associated with several kinds of human autoimmune diseases, suggesting that CTLA-4 gene is probably a general susceptibility gene to autoimmune disease. The present study was conducted in Chinese to determine whether there is any association of the CTLA-4 gene polymorphism with the development of systemic lupus erythematosus (SLE). CTLA-4 gene polymorphism in promoter and exon 1 was detected by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method in 81 patients with SLE and 81 normal controls. The results showed that there were no statistically significant differences in both exon 1 and promoter gene polymorphism between SLE patients and normal controls. The preliminary study does not suggest an association of the known polymorphism in exon 1 and promoter of CTLA-4 gene with Chinese SLE. However, SLE is a very heterogeneous syndrome and CTLA-4 gene polymorphism might correlate with some specific clinical features. To exploring this possibility, subgroup analysis in more patients needs to be performed.

Decreased CD4+CD25+bright T cells in peripheral blood of patients with primary Sjögren's syndrome

CD4+CD25 +bright T cells played a crucial role in the suppression of immune response. Recently, decreased levels of CD4+CD25+bright T cells in the peripheral blood of patients with systemic lupus erythematosus were reported, suggesting the potential role of CD4+CD25+bright T cells in human autoimmune diseases. Primary Sjögrens syndrome (pSS) is another common human systemic autoimmune disease. The present study aimed to investigate the levels of CD4+CD25+bright T cells in pSS and to correlate their levels with some biomarkers of inflammation and immune activation. Thirty-three patients with pSS and 35 age- and sex-matched normal individuals were enrolled in the study. The flowcytometric method was applied in the measurement of CD4+CD25+bright T cells. The results showed that patients with pSS had statistically lower levels of CD4+CD25+bright T cells than normal controls, expressed either as absolute cell numbers (mean ± SD: 47.07 ± 25.53 cells/mm 3 versus 79.55 ± 34.56 cells/mm3, P < 0.001) or as percentages of peripheral blood mononuclear cells (mean ± SD: 2.79 ± 1.06% versus 3.84 ± 1.42%, P < 0.001) or as percentages of CD4+ T cells (mean ± SD: 7.85 ± 2.62% versus 11.68 ± 3.78%, P < 0.005). Moreover, there were statistically significant inverse correlations between the levels of CD4+CD25+bright T cells and some parameters of inflammation or immune activation including erythrocyte sedimentation rate, C-reactive protein, IgG and rheumatoid factors. The result suggested that CD4+CD25+bright T cells were likely to play anti-inflammatory and immunosuppressive roles in the pathogenesis of pSS. However, the exact functions of decreased circulating CD4+CD25+bright T cells in pSS need further elucidated. Lupus (2008) 17, 34—39.