Chien-Hsueh Tung

Higher LPS-stimulated TNF-α mRNA levels in peripheral blood mononuclear cells from Chinese ankylosing spondylitis patients with −308G/A polymorphism in promoter region of tumor necrosis factor: association with distinct A33/B58/Cw10 haplotypes

To investigate the effects of TNF-α −308, −238 promoter polymorphisms on TNF-α transcription in B27 positive Chinese patients with ankylosing spondylitis (AS). The possible relationship between polymorphisms, MHC antigens, and quantitative TNF-α mRNA expression were evaluated. Single nucleotide polymorphisms (SNPS) of TNF-α −308 and −238 were performed by PCR-amplification refractory mutation system method (PCR-ARMS) in sixty-seven B27-positive AS patients and 60 HLA-B27 positive healthy controls in Chinese. Quantitative measurement of TNF-α mRNA in peripheral blood mononuclear cells was performed with real time RT-PCR. The polymorphisms were correlated to quantitative TNF-α mRNA, and MHC antigens (determined by SSP method) in AS patients. The prevalence rate of both −308G/A and −238G/A TNF-α promoter polymorphisms in patients were not significantly different from those in normal subjects. However, a significant high LPS-stimulated TNF-α mRNA expression was found in peripheral blood mononuclear cells from patients with promoter −308G/A polymorphism (TNF2) as compared to those in −308G/G genotype (TNF1). Furthermore, −308G/A polymorphism in patients was found to be tightly associated with distinct haplotypes of A33/B58/Cw10 [12 out of 14 –308G/A patients (85.7%) versus none in 53 –308G/G patients], independent of B27 antigen. HLA-A33-B58-Cw10 haplotypes associated TNF-α promoter −308G/A polymorphism might play an important role in disease pathogenesis of AS in Chinese population, partially related to a driving force of a higher TNF-α production. It confirms once again the importance and complexity of MHC related molecules in disease pathogenesis of AS.

Increased expression of long noncoding RNAs LOC100652951 and LOC100506036 in T cells from patients with rheumatoid arthritis facilitates the inflammatory responses.

The aim of this study was to evaluate whether the presence of aberrantly expressed lncRNAs could promote T cell inflammatory responses in patients with RA. The expression levels of 10 potential aberrantly expressed lncRNAs were evaluated in T cells from 39 patients with RA and 17 controls using real-time reverse transcription polymerase chain reaction. The aberrantly expressed lncRNAs were measured in Jurkat cells co-cultured with or without ionomycin and phorbol 12-myristate 13-acetate. Transfection studies using small interfering RNA (siRNA) were conducted for biological functions, and microarray analysis was performed to search for target genes of specific lncRNAs. We confirmed that the expression levels of LOC100652951 and LOC100506036 were higher in RA T cells compared with controls. RA patients treated with biologic agents had lower expression levels of LOC100652951, and female RA patients had lower LOC100506036 expression levels after multivariate analysis. After activation, the expression levels of LOC100506036, but not LOC100652951, increased in Jurkat cells. Transfection of siRNA targeting LOC100506036 inhibited interferon gamma production and the expression of nuclear factor of activated T cells in activated Jurkat cells. After the microarray analysis with validation, inhibition of LOC100506036 expression by siRNA leaded to the decreased expression of sphingomyelin phosphodiesterase 1 (SMPD1). In conclusion, the expression levels of LOC100652951 and LOC100506036 were increased in RA T cells. Treatment with biologic agents could lower the expression of LOC100652951 in RA T cells. LOC100506036 could regulate the expression of SMPD1 and NFAT1 and could contribute to the inflammatory responses in RA.

Anti-citrullinated protein antibodies activated ERK1/2 and JNK mitogen-activated protein kinases via binding to surface-expressed citrullinated GRP78 on mononuclear cells.

In a previous study, we found that anti-citrullinated protein antibodies (ACPAs) enhance nuclear factor (NF)-κB activity and tumor necrosis factor (TNF)-α production by normal human peripheral blood mononuclear cells (PBMCs) and U937 cells via binding to surface-expressed citrullinated glucose-regulated protein 78 (cit-GRP78). However, the downstream signaling pathways remain unclear after binding. In the present study, we firstly measured the effects of different kinase inhibitors on ACPA-mediated TNF-α production from normal PBMCs and monocytes. Then, the native and phosphorylated mitogen-activated protein kinases (MAPKs) were detected in ACPA-activated U937 cells by Western blotting. We also explored the role of the phosphoinositide 3-kinase (PI3K)-Akt pathway in activating IκB kinase alpha (IKK-α) in ACPA-stimulated U937 cells. Finally, we measured the amount of cit-GRP78 from PBMC membrane extracts in RA patients and controls. We found that MAPK and Akt inhibitors, but not PI3K inhibitor, remarkably suppressed ACPA-mediated TNF-α production. Interestingly, ACPAs selectively activated extracellular signal-regulated kinase 1/2 (ERK1/2) and c-jun N-terminal kinase (JNK), but not p38 MAPK, in U937 cells. This activation was suppressed by cit-GRP78, but not GRP78. The JNK activation further enhanced the phosphorylation of Akt and IKK-α. The expression of cit-GRP78 on cell membrane was higher in RA than normal PBMCs. Taken together; these results suggest that through binding to surface, over-expressed cit-GRP78 on RA PBMCs, ACPAs selectively activate ERK1/2 and JNK signaling pathways to enhance IKK-α phosphorylation, which leads to the activation of NF-κB and the production of TNF-α .

Increased prevalence of polyomavirus BK viruria that correlates with thrombocytopenia in patients with systemic lupus erythematosus on intensive immunosuppressive therapy

The prevalence of polyomavirus BK (BKV) reactivation is high in patients with systemic lupus erythematosus (SLE) on long-term immunosuppressants compared to normal population. However, only a few studies are available for the possible correlation of BKV reactivation and clinical manifestations in SLE patients. In the present study, we tried to correlate BKV viruria, clinical manifestations, laboratory findings, and medications in patients with SLE. The urine BKV viral DNA copies were detected from 95 patients with SLE and 32 healthy volunteers by real-time PCR. We found that the prevalence rate of BKV viruria in SLE patients was significantly higher than normal group (71.6% vs. 18.6%, p < 0.001) as well as the urine BKV DNA viral load (4.74 ± 3.17 vs. 1.08 ± 2.33 by log scale, p < 0.001). Interestingly, BKV viruria (+) SLE patients had more thrombocytopenic events than BKV viruria ( − ) group (32.4% vs. 3.7%, p = 0.008 after adjustment by age and sex). The patients with BKV viruria DNA copy number >3,200,000/ml exhibited more thrombocytopenia risk than BKV viruria ≦3,200,000 copy number/ml or BKV viruria ( − ). The use of potent immunosuppressants may increase BKV viruria. In a refractory thrombocytoponeic case, the add-on of anti-BKV medication, leflunomide 20 mg/day rapidly decreased BKV viruria and recovered platelet counts. In conclusion, our study demonstrated that patients with SLE had higher prevalence rate of BKV reactivation that is correlated with thrombocytopenic episode. Intensive immunosuppressive therapy in SLE may increase the risk of BKV viruria.

Nifedipine suppresses Th1/Th2 cytokine production and increased apoptosis of anti-CD3 + anti-CD28-activated mononuclear cells from patients with systemic lupus erythematosus via calcineurin pathway

Increased Ca(2+) influx is found in mononuclear cells (MNC) of patients with systemic lupus erythematosus (SLE). The role of calcineurin and potential implication of calcium channel blocker to suppress the abnormal Ca(2+) influx in SLE remain to be determined. In the present study, we found that the expression and phosphatase activity of calcineurin, but not calcineurin inhibitor in SLE-MNC were greater than normal MNC. Functionally, 1 microM nifedipine could suppress SLE-MNC IFN-gamma secretion but 10 microM nifedipine was required for suppressing that of normal MNC. IL-10 secretion by both SLE-MNC and normal MNC was suppressed by 1 microM nifedipine. However, high dose of nifedipine (50 microM) suppressed NFATc1 activation in SLE-MNC and enhanced apoptosis of anti-CD3 + anti-CD28-activated SLE-MNC irrelevant to expression of Fas ligand. These data suggest that SLE-MNC overexpressed calcineurin and hyper-responded to L-type Ca(2+) channel blocker-mediated apoptosis and cytokine suppression. We proposed that L-type Ca(2+) channel blocker maybe a potential medication for controlling SLE.

Patterns of Ambulatory Medical Care Utilization and Rheumatologist Consultation Predating the Diagnosis of Systemic Lupus Erythematosus: A National Population-Based Study

Objective To investigate the records of ambulatory medical care from patients predating the diagnosis of systemic lupus erythematosus (SLE) using nationwide, population-based claims data. Methods The frequencies and costs of ambulatory medical care utilization in 337 newly-diagnosed SLE cases between 2004 and 2010, identified from Taiwans National Health Insurance Research Database, were compared with 1,348 controls who were frequency matched for sex, age, and the catastrophic illness certificate application year of the cases. Results Patients with SLE had a median frequency of ambulatory medical care utilization compared with controls one year prior to the index date (22 vs. 2, P<0.001). The differences were significant throughout all eight annual periods. Similarly, the inflation-adjusted costs of ambulatory medical care utilization in patients with SLE increased annually over the study period, from a median of US

HLA Haplotype A33-B58-Cw10 May Modulate Radiographic Development of Bamboo Spine in Taiwanese Patients with Primary Ankylosing Spondylitis

18 eight years prior to the index date to US

Tumor necrosis factor-α blockade treatment decreased CD154 (CD40-ligand) expression in rheumatoid arthritis

680 one year prior to the index date. Diseases of the respiratory system (International Classification of Diseases, Ninth Revision, Clinical Modification [ICD-9-CM] codes 460–519), digestive system (ICD-9-CM codes 520–579), musculoskeletal system and connective tissue (ICD-9-CM codes 710–739, excluding 710.0), and skin and subcutaneous tissue (ICD-9-CM codes 680–709) were the top four common causes of visits in the 0.5 to 2 year period preceding the index date and percentages of SLE patients suffered from these disorders increased progressively over the study period. Only 56.4% of the patients with SLE had consulted a rheumatologist and most of the serology tests were done within one year predating the index date. Conclusions Increased frequencies and costs of ambulatory care utilization among Taiwanese patients with SLE occurred several years predating their definitive SLE diagnosis. When multisystemic disorders are presented in young female patients, the possibility of SLE should be considered and screened with tools such as the antinuclear antibody test.

Incident osteoarthritis and osteoarthritis-related joint replacement surgery in patients with ankylosing spondylitis: A secondary cohort analysis of a nationwide, population-based health claims database

Objective: To evaluate the possible relationship between HLA alleles and bony ankylosis of the spine (bamboo spine) in Taiwanese patients with ankylosing spondylitis (AS). Methods: A small cohort of HLA-B27 positive AS patients was conducted to analyze the effects of alleles and haplotypes on the development of bamboo spine. DNA typing of HLA class I and class II genes were performed by SSP method on primary ankylosing spondylitis patients with bamboo spine (n = 84) and spinal enthesopathy controls (n = 228). Odds ratios with 95% confidence intervals and P value were estimated. Determination of the most probable HLA haplotypes on all patients were constructed by comparison of the alleles carried by each patient with the HLA haplotype database established in Taiwanese population studies using homozygosity approach [1] and by expectation-maximum algorithm [2]. Results: Allele frequencies of HLA A33, B58, Cw10, DR4, DR17 and DQ2 were significantly lower in bamboo patients as compared to non-bamboo controls. In contrast, allele frequencies of A24, B54, Cw15, DR11 and DR14 were significantly higher in bamboo patients. Less remarkably, high frequencies of B39, B51, Cw1 and Cw2 alleles were also noted in bamboo patients. Considering linkage disequilibria of alleles in haplotypes, HLA-A11-B27-Cw12 was the most common haplotype in both bamboo and non-bamboo groups (95.23% vs. 91.22%, respectively, P = 0.238). Haplotypes A33-B58-Cw10, A33-B58-Cw10-DR13 and A33-B58-Cw10-DR17 were significantly lower in bamboo patients as compared to those in controls (P < 0.001, P = 0.001, P = 0.002, respectively). Conclusion: Haplotypes A33-B58-Cw10, A33-B58-Cw10-DR13 and A33-B58-Cw10-DR17 showed a strong association with bamboo spine in Taiwanese AS patients. Detection of such haplotypes might be a great aid in the management of patients with the disease.

Increased prevalence of JC polyomavirus viruria was associated with arthritis/arthralgia in patients with systemic lupus erythematosus.

Contexts CD154 (commonly referred to as CD40-ligand) is a critical T cell factor that participates in the pathogenesis of autoimmune and is over-expressed in rheumatoid arthritis (RA). TNF-α blockade treatment had dramatic efficacy in RA. Objective To investigate whether TNF-α blockade treatment can inhibit CD154 expression in RA. Methods Blood samples were collected from 33 patients with rheumatoid arthritis before and 3 months after TNF-α blockade treatment. Clinical serological data determined by standard assays and T cell CD154 expression levels determined by flow cytometry were statistically analyzed for these two time points. Results The percentage of CD154 expression on gated CD4+ T cells of PBMCs from RA patients after 3 months TNF-α blockade treatment was significantly lower than before treatment (2.94 ± 3.21% vs. 7.21 ± 5.64%; p = 0.0001). The disease activity and anti-CCP antibody levels were also significantly reduced after TNF-α blockade treatment. The CD154 expression levels were positively correlated with disease activity index DAS28, and CRP. The post-stimulated CD154 expression percentage of purified CD4+ T cells between baseline and after TNF-α blockade treatment was not significantly different (p = 0.221). Baseline CD154 levels were positively correlated with treatment-induced changes in DAS28 (p = 0.014; r2 = 0.187). Conclusions TNF-α blockade treatment significantly decreased the CD154 expression on CD4+ T cells, disease activity and anti-CCP antibody simultaneously in RA patients. However TNF-α blockade did not impair T cell capacity to express CD154 after stimulation. These results suggest that decreased CD154 expression after TNF-α blockade may be due to decreased RA disease activity but not direct inhibition of CD154 responsiveness of T cells.