Chang-Youh Tsai

Increased Excretions of β2-Microglobulin, IL-6, and IL-8 and Decreased Excretion of Tamm-Horsfall Glycoprotein in Urine of Patients with Active Lupus nephritis

Tubulointerstitial nephritis is a less frequently recognized but important complication of systemic lupus erythematosus. We have investigated the cytokine β2-microglobulin (β2M) and Tamm-Horsfall glycoprotein (THG) excretions in the urine of systemic lupus erythematosus patients to identify indices for evaluation of tubulointerstitial inflammation in lupus nephritis (LN). Daily urine was collected from 15 patients with active LN, from 12 patients with inactive LN, and from 17 normal subjects. The amounts of soluble interleukin (IL) 2 receptor, IL-6, IL-8, β2M, and THG in urine were measured. β2M and THG were regarded as indicators of proximal and distal renal tubule function, respectively. The urinary excretions of IL-6 and IL-8 were significantly higher in patients with active LN than in those with inactive LN and in normal individuals. The excretion of soluble IL-2 receptor in all three groups of subjects was not significantly different. On the other hand, the excretion of β2M in patients with LN was significantly higher than that in normal individuals. The excretion of β2M in patients with active or inactive LN was not significantly different. The THG excretion was lower in patients with active LN and tubulointerstitial inflammation as compared with patients with inactive LN or normal individuals. Six patients underwent pulse cyclophosphamide therapy during the course of experiments. Five of them showed a decrease in IL-8 and IL-6 excretions in urine after the treatment. The excretions of β2M and THG in urine, in addition to IL-6 and IL-8, can reflect the renal inflammatory activity in patients with lupus tubulointerstitial nephritis as well as in those having lupus glomerulonephritis.

The effect of etanercept on anti‐cyclic citrullinated peptide antibodies and rheumatoid factor in patients with rheumatoid arthritis

Objective: To evaluate the changes in anti-cyclic citrullinated peptide antibodies (anti-CCP) and rheumatoid factor (RF) following etanercept treatment in patients with rheumatoid arthritis. Methods: The study included 90 patients with rheumatoid arthritis who failed treatment with disease modifying antirheumatic drugs (DMARDs). All patients were allowed to continue treatment with DMARDs; 52 of them received etanercept as a twice weekly 25 mg subcutaneous injection for three months, and the others did not. Serum samples were collected at baseline and one month intervals during the treatment course. The serum levels of anti-CCP and RF were tested by enzyme linked immunosorbent assay and nephelometry, respectively. Results: At baseline, 45 of the 52 etanercept treated patients (86.5%) and 32 of the 38 controls (84.2%) were positive for anti-CCP. Tests for RF were positive in 78.9% and 84.2% of patients with or without etanercept treatment, respectively. The serum levels of anti-CCP and RF decreased significantly after a three month etanercept treatment (p = 0.007 and p = 0.006, respectively). The average decrease from baseline calculated for each individual patient in the etanercept treated group was 31.3% for anti-CCP and 36% for RF. The variation in anti-CCP was positively correlated with the variation in disease activity, swollen and tender joint counts, RF, and C reactive protein. Conclusions: Etanercept combined with DMARDs leads to a much greater decrease than DMARDs alone in the serum levels of anti-CCP and RF in rheumatoid arthritis, compatible with a reduction in clinical disease activity.

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.

Association of polymorphisms in complement component C3 gene with susceptibility to systemic lupus erythematosus

OBJECTIVE Identification of the genes responsible for systemic lupus erythematosus (SLE). METHODS All the exons and putative promoter regions of 53 candidate genes (TNFRSF6/Fas, TNFSF6/FasL, Fli1, TNFSF10/TRAIL, TNFSF12/TWEAK, Bcl-2, PTEN, FADD, TRADD, CDKN1A, TNFRSF1A/TNFR1, TNFRSF4/OX40, TNFSF4/OX40L, TNFSF5/CD40L, TNFSF13B/BAFF, ICOS, CTLA4, CD28, FYN, G2A, CR2, PTPRC/CD45, CD22, CD19, Lyn, PDCD1, PTPN6, TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, TGFBR3, CD3Z, DNASE1, APCS, MERTK, C3, C1QA, C1QB, C1QG, C2, MBL2, IGHM, IL-2, IL-4, IL-10, IFNG, TNFA, MAN2A1, TNFRSF11A/RANK, TNFRSF11B/OPG, TNFSF11/OPGL) were screened for single nucleotide polymorphisms (SNPs) and their association with SLE was assessed by case-control studies. A total of 509 cases and 964 controls of Japanese descent were enrolled. RESULTS A total of 316 SNPs was identified. When analysed in the Japanese population, the allele frequencies of T at rs7951 and G at rs2230201 of the C3 gene were 0.110 and 0.626, respectively, in SLE patients; significantly higher than the frequencies of 0.081 and 0.584, respectively, in controls [odds ratio (OR) = 1.40, 95% confidence interval (CI) = 1.05-1.86, P = 0.016 and OR=1.19, 95% CI = 1.01-1.41, P = 0.038, respectively]. The mean serum C3 level of carriers of the rs7951 T allele was significantly lower than that of non-carriers of the T allele in 87 SLE patients whose medical records were available (P = 0.0018). CONCLUSION rs7951 T allele of the C3 gene was significantly associated with SLE, and decreased serum level of C3 seems to be correlated with this allele.

Increased Excretion of Tumor Necrosis Factor Alpha and Interleukin 1 β in Urine from Patients with IgA Nephropathy and Schönlein-Henoch Purpura

Urinary proteins (5 mg/ml) collected from a group of 16 patients including 13 with IgA nephropathy and 3 with Schönlein-Henoch purpura (SHP) and from a control group consisting of 6 patients with diabetic nephropathy, 5 patients with hypertensive nephrosclerosis, and 5 healthy hospital staff members were studied for the contents of interleukins (IL) 1 beta, 2, 4, 6, and 12 and tumor necrosis factor alpha (TNF-alpha). Eleven patient with IgA nephropathy or SHP (11/16) but only 1 of the controls (1/16) had TNF-alpha activity in urinary proteins (p < 0.01). The IL-1 beta activity exhibited a similar tendency but to a lesser extent (10 of 16 patients with IgA nephropathy or SHP vs. 2 of 16 with other conditions, p < 0.05). Conversely, the detection rates of IL-2, IL-4, and IL-6 in both groups were not significantly different. IL-12 was not found in any of the samples from both groups. Sera and nonpurified urine samples from the same individuals were also measured for cytokines. IL-1 beta, IL-2, IL-4, and IL-12 were absent in all these samples, but TNF-alpha was found in four of the serum samples from patients with IgA nephropathy. Urinary proteins (2 mg/ml) were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereby peptides of 52, 49, 45, 34, 30, and 11 kD could be demonstrated in the patients with IgA nephropathy or SHP. Urinary proteins (200 micrograms/ml from patients with IgA nephropathy or SHP exerted a mitogen-like effect on the normal human mononuclear cells, as demonstrated by 3H-thymidine incorporation. In addition, these urinary proteins (400 micrograms/ml) enhanced the proliferative activity of the cultured rat glomerular mesangial cells. The exaggerated proliferation of rat glomerular mesangial cells exerted by urine proteins from 2 patients with active disease was markedly suppressed after treatment with glucocorticoids/cyclophosphamide. These results suggest that patients with IgA nephropathy or SHP can excrete excessive amounts of TNF-alpha and IL-1 beta in the urine. The inconsistent presence of these two cytokines in urine and serum may indicate that they can be produced locally and that they are implicated in the development of mesangial inflammation and glomerular damage.

Serum BLC/CXCL13 Concentrations and Renal Expression of CXCL13/CXCR5 in Patients with Systemic Lupus Erythematosus and Lupus Nephritis

Objective. Systemic lupus erythematosus (SLE) is a prototype of systemic autoimmune disease in which cytokines such as B lymphocyte chemoattractant (BLC, or CXC motif ligand 13, CXCL13) may play important roles in pathogenesis. We investigated the implications of CXCL13 in SLE and lupus nephritis. Methods. Serum samples from 425 patients with SLE and 106 healthy control individuals were analyzed for the concentration of CXCL13 by ELISA. Tissue expression of CXCL13 and its corresponding receptor CXCR5 were observed in lupus kidney. The CXCR5-bearing B cells in SLE patients were analyzed by flow cytometry. Results. Serum levels of CXCL13 were higher in SLE patients compared to controls. SLE patients with lupus nephritis or positive anti-dsDNA antibodies had significantly higher serum CXCL13 levels. The peripheral venous blood B cells that bear CXCR5 were more abundant in SLE patients as detected by flow cytometry. CXCR5 and CXCL13 were highly expressed in the renal cortex from patients with lupus nephritis. Conclusions. Our results suggest that BLC/CXCL13 as well as its corresponding receptor, CXCR5, may play important roles in the pathogenesis of SLE and in lupus nephritis.

Association of serum interleukin-17 and interleukin-23 levels with disease activity in Chinese patients with ankylosing spondylitis.

Background: Ankylosing spondylitis (AS) is a chronic arthritis with a pathogenesis which is not fully understood. A third subset of IL‐17‐producing T helper cells, called Th17 cells, has been discovered and characterized. We investigated whether IL‐17 and IL‐23, two Th17‐related cytokines, play any roles in the pathogenesis of, and have any correlations with, disease activity and clinical manifestations in AS. Methods: This cross‐sectional study included 49 AS patients and 25 healthy control subjects. The serum IL‐17 and IL‐23 levels were measured using enzyme‐linked immunosorbent assay kits. At the same time, C‐reactive protein (CRP), erythrocyte sedimentation rate (ESR), Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI) and Bath Ankylosing Spondylitis Patient Global Score (BAS‐G) levels were measured, and physical examinations were performed on study participants to determine their extent of physical mobility. Results: The serum IL‐17 and IL‐23 levels of the AS patients were significantly higher than those of the healthy controls. In the AS patients, the BASDAI scores had a better correlation with the serum IL‐17 or IL‐23 levels (IL‐17, r = 0.351, p = 0.014; IL‐23, r = 0.398, p = 0.005) than with ESR (r = 0.078, p = 0.600) and CRP (r = 0.012, p = 0.993). IL‐17 or IL‐23 correlate to the BASFI, BAS‐G and parameters related to physical mobility. In the receiver operating characteristic (ROC) analysis, the serum IL‐17 and IL‐23 levels act better in discriminating patients with BASDAI≥4 (AUC value 0.88, p = 0.001) than ESR and CRP (AUC value 0.727, p = 0.008). Conclusion: Serum IL‐17 and IL‐23 levels were significantly higher in AS patients than in healthy controls and the levels correlate to disease activity measured by BASDAI scores, but not parameters of functional ability and spinal mobility. These results suggest the existence of a role of IL‐17 and IL‐23 in the pathogenesis of inflammation in AS.

Spontaneous resolution of acute gouty arthritis is associated with rapid induction of the anti-inflammatory factors TGFβ1, IL-10 and soluble TNF receptors and the intracellular cytokine negative regulators CIS and SOCS3

Objective The molecular basis for spontaneous resolution of acute gouty arthritis (GA) remains unclear. The hypothesis that extracellular and intracellular mechanisms play roles in resolving acute GA was tested. Methods Synovial fluid (SF) levels of transforming growth factor β1 (TGFβ1), interleukin 1 (IL-1) receptor antagonist (IL-1ra), IL-10 and soluble tumour necrosis factor (TNF) receptor I (sTNFRI) and II (sTNFRII) were measured by ELISA in patients with acute GA and osteoarthritis (OA). Monosodium urate (MSU) crystal-stimulated RAW264.7 mouse macrophages were analysed for cytokine inducible SH2-containing protein (CIS) and suppressors of cytokine signalling (SOCS)1-7 mRNA expression by reverse transcription (RT)-PCR. Immunohistochemical analysis, quantitative PCR and immunoblotting were performed to detect CIS and SOCS3 expression in synovial tissue, SF mononuclear cells (SFMCs) from patients with GA and MSU crystal-stimulated monocyte-derived macrophages from healthy donors. CIS overexpression and small interfering RNA-mediated knockdown in RAW264.7 cells were used to investigate the role of CIS in resolving MSU crystal-induced acute inflammation. Results SF levels of anti-inflammatory molecules TGFβ1, IL-1ra, IL-10 and sTNFR-I/II were significantly elevated in GA compared to OA. CIS and SOCS3 were upregulated in the synovium and SFMCs from acute GA and MSU crystal-stimulated monocyte-derived macrophages and RAW264.7 cells. CIS overexpression in RAW264.7 cells attenuated MSU crystal-induced IL-1β and TNFα but enhanced TGFβ1 production via increased binding of signal transducer and activator of transcription 3 (STAT3) to the TGFβ1 promoter. Conversely, CIS knockdown reversed the effect of CIS overexpression, resulting in enhanced IL-1β and TNFα but reduced TGFβ1 production in MSU crystal-stimulated RAW264.7 cells. Conclusions Increased production of TGFβ1, IL-1ra, IL-10 and sTNFR-I/II and upregulation of intracellular CIS and SOCS3 expression are associated with spontaneous resolution of acute GA.

Tamm-Horsfall urinary glycoprotein enhances monokine release and augments lymphocyte proliferation.

Tamm-Horsfall glycoprotein (THG) purified from pregnancy urine was found to stimulate normal human mononuclear cell (MNC) proliferation at a concentration greater than 10 micrograms/ml. This stimulation was non-specific because the percentage of B and T cell subpopulations including CD20, CD3, CD4, CD8 and CD4/CD8 ratio was not changed by THG. THG not only bound to human mononuclear cells but depolarized the membrane potential, increased 22Na+ uptake and enhanced the expression of IL-2R and HLA-class II antigens on these cells. The concentrations of sIL-2R, sCD4 and sCD8 in the THG-stimulated MNC culture supernatants were significantly increased compared with control supernatants. In addition, overnight incubation of THG (5-50 micrograms/ml) with MNC dose-responsively enhanced the syntheses of IL-1 beta, IL-6 and TNF-alpha by monocytes, with a maximal effect at 25 micrograms/ml. This monokine releasing activity of THG could be neutralized by a specific antibody against THG. When monocytes/macrophages were depleted from mononuclear cells by incubating with lysosomotropic methyl ester of L-leucine, THG retained the capability of stimulating lymphocytes proliferation but to a lesser degree. These results suggest that urinary THG activates monocytes to synthesize large amount of monokines through its membrane effect. The released monokines subsequently stimulate lymphocytes expressing IL-2R and HLA-class II antigens and finally lead to cell proliferation.

Ectopic and high CXCL13 chemokine expression in myasthenia gravis with thymic lymphoid hyperplasia.

Myasthenia gravis (MG) is an antibody and complement mediated autoimmune disease. Serum CXC chemokine ligand 13 (CXCL13) was found to be elevated in MG patients and high CXCL13 level was associated with severe clinical stages, especially in females with thymic lymphoid hyperplasia. Both protein and mRNA of CXCL13 and CXC chemokine receptor 5 (CXCR5) in the thymic tissues were significantly higher in MG patients with lymphoid hyperplasia than those with thymoma. Our data indicated that serum CXCL13 can be used as an index of disease severity and ectopic thymic expression of CXCL13 might be associated with aberrant cell trafficking to the thymus of MG.