Tsai-Ching Hsu

Elevated circulatory MMP-2 and MMP-9 levels and activities in patients with rheumatoid arthritis and systemic lupus erythematosus

OBJECTIVES Matrix metalloproteinases (MMPs) are suggested to play important roles in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). This study is to examine the MMPs expressions and activities in Taiwanese RA and SLE patients. DESIGN AND METHODS Levels and activities of plasma MMP-2 and MMP-9 were investigated by enzyme-linked immunosorbent assay and zymography, respectively. RESULTS MMP-2 levels in control subjects, RA and SLE patients were 146.1+/-34.2, 194.0+/-24.2 and 208.9+/-75.9 ng/mL respectively, and for MMP-9 were 51.4+/-57.1, 567.7+/-313.1 and 208.7+/-105.5 ng/mL respectively. Both MMP-2 and MMP-9 levels and activities from all patients were significantly higher than that from control subjects. CONCLUSIONS MMP-2 levels in both patients groups were approximately 1.3-1.4 folds higher than that in control subjects, notably, MMP-9 levels were 11- and 4-folds significantly higher, respectively, in RA and SLE patients. The results which MMP-2 and MMP-9 levels and activities are significantly elevated support the involvement of MMPs proteins in these autoimmune disorders.

Proteinase 3 and dihydrolipoamide dehydrogenase (E3) are major autoantigens in hepatitis C virus (HCV) infection.

Hepatitis C virus (HCV) infection has been found to be strikingly associated with autoimmune phenomena. The aim of the present study was to investigate the presence of various autoantibodies in patients with HCV infection. Anti‐neutrophil cytoplamic antibody (ANCA), anti‐dihydrolipoamide dehydrogenase (anti‐E3), rheumatoid factor (RF), anti‐dihydrolipoamide acetyltransferase (anti‐E2), anti‐SS‐A/Ro (60 kD), anti‐SS‐A/Ro (52 kD), anti‐SS‐B/La, anti‐topoisomerase II (anti‐topo II), anti‐cardiolipin (aCL), anti‐dsDNA, anti‐ssDNA, anti‐nuclear antibodies (ANA), anti‐proteinase 3 (anti‐Pr3) and anti‐myeloperoxidase (anti‐MPO) were determined in sera from 516 patients with HCV infection, 11 with primary biliary cirrhosis (PBC) and 44 healthy controls. Assays employed were indirect immunofluoresence, the particle latex agglutination test, enzyme‐linked immunosorbent assay (ELISA) and immunoblotting. ANCA, anti‐E3 antibody and RF were positive in 278/516 (55·6%), 276/516 (53·3%) and 288/516 (56%) patients with HCV infection, respectively. Positivity for ANA was present in 15·8%, anti‐ssDNA in 15·6%, anti‐dsDNA in 8·5%, aCL in 5%, anti‐SS‐B/La in 4·1%, anti‐SS‐A/Ro (60 kD) in 3·9%, anti‐E2 in 3·3% and anti‐SSA/Ro (52 kD) in 1·2%, anti‐MPO in 4·8%, anti‐Topo II and anti‐actinin in 0%. All sera with ANCA showed c‐ANCA patterns and contained anti‐PR3 specificity. HCV patients with ANCA showed a higher prevalence of skin involvement, anaemia, abnormal liver function and α‐Fetoprotein (α‐FP). HCV patients with anti‐E3 antibodies showed a higher prevalence of liver cirrhosis, arthritis, abnormal liver function and elevated α‐FP levels. The prevalence of autoantibodies was not affected by treatment with interferon‐alpha (IFN‐α). In conclusion, autoantibodies are commonly found in patients with HCV infection. There is a high prevalence of anti‐E3, ANCA and RF in these patients. Proteinase 3 and E3 are the major target antigens in HCV infection. HCV may be regarded as a possible causative factor in ANCA‐related vasculitis.

Expression of anti-cardiolipin antibodies and inflammatory associated factors in patients with schizophrenia

Numerous studies have implicated a connection between schizophrenia and autoimmune disorders. However, the precise relationship and underlying mechanism are still obscure. To further identify the association between autoimmune disorders and schizophrenia, the mRNA expressions of various cytokines and Toll-like receptors (TLRs) in monocytes are examined by using RT-PCR. Additionally, ELISA and zymography were performed to determine the anti-cardiolipin antibody (aCL) and MMP9 activity in serum form schizophrenic patients. Notably, significantly increased interleukin (IL)-6 and IL-10 mRNA were observed in schizophrenic patients, whereas significant reductions of TLR-3 and TLR-5 mRNA were detected. Moreover, significantly increased levels of aCL antibody and a higher frequency of positive-MMP9 activity were detected in serum from patients with schizophrenia. Meanwhile, no significant association was found between each of the medications and aCL activity. These findings demonstrated autoimmune-related phenomena in schizophrenic patients and further suggested a connection between schizophrenia and autoimmune disorders.

Human parvovirus B19 non-structural protein (NS1) induces apoptosis through mitochondria cell death pathway in COS-7 cells

Human parvovirus B19 has been found in various tissues in addition to erythroid lineage cells, and non-structural protein (NS1) is reported to induce cytotoxicity and apoptosis in erythroid lineage cells, but the mechanism in non-permissive cells is still unclear. To address this issue, we have constructed the NS1 gene in a cytomegalovirus episomal vector, pEGFP-C1 and transfected it into monkey epithelial cells, COS-7. EGFP-NS1 expression in transfected cells was monitored and assessed by fluorescence microscopy, RT-PCR and Western blot. The flow cytometric analysis showed that the NS1-transfected cells were arrested at G1 phase by paclitaxel treatment and there was increased apoptosis. The expression of p53, an important molecule in apoptosis and cell cycle regulation, and its downstream cell cycle kinase inhibitors p16INK4 and p21WAF1/CIP1 were up-regulated in the NS1-transfected cells. Also, increased expression of the pro-apoptotic Bcl-2 members Bax, Bad and activation of caspase 3 and caspase 9, but not the activation of caspase 8 or Fas were detected in the NS1-transfected cells. p53-induced Bax expression and subsequent activation of caspase 9 is probably the apoptotic pathway in NS1-transfected cells since activation of the caspase 9 was suppressed by the p53 inhibitor and apoptosis was significantly inhibited by the caspase 9 inhibitor. Our results suggest that the cell death of the NS1-transfected cells is associated with mitochondria related apoptosis. These findings might provide alternative information for further study and characterization of B19 NS1 protein in B19 non-permissive cells.

Increased expression and secretion of interleukin-6 in human parvovirus B19 non-structural protein (NS1) transfected COS-7 epithelial cells

Human parvovirus B19 (B19) has been associated with a variety of autoimmune diseases, including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We have demonstrated previously that B19 non‐structural protein (NS1) induced apoptosis through the mitochondria cell death pathway in COS‐7 epithelial cells and that B19 NS1 may play a role in the pathogenesis of autoimmune diseases. In order to examine the expression profiles of cytokines and chemokines in B19 NS1 transfected COS‐7 cells, we constructed the NS1 gene in the pEGFP‐C1 vector named enhanced green fluorescence protein gene (EGFP)‐NS1. COS‐7 cells were transfected with EGFP or EGFP‐NS1 plasmid. The expression profiles of cytokines and chemokines, including interleukin (IL)‐1β, IL‐5, IL‐6, IL‐8, IL‐10, tumour necrosis factor (TNF)‐α, transforming growth factor (TGF)‐β, granulocyte–macrophage colony‐stimulating factor (GM‐CSF), growth‐related oncogene α (GROα), interferon gamma‐inducible protein (IP)‐10, stromal cell derived factor (SDF)‐1, macrophage inflammatory protein (MIP)‐1β, monocyte chemoattractant protein (MCP)‐1, regulated upon activation normal T cell expressed and secreted (RANTES), Fractalkine, CX3CR1, CCR2, CCR5 and CCR11 were examined in COS‐7 cells, EGFP and EGFP‐NS1 transfected cells using enzyme‐linked immunosorbent assay (ELISA) or reverse transcription–polymerase chain reaction (RT–PCR). Increased expression and levels of IL‐6 were found in EGFP‐NS1 transfected cells using RT–PCR and ELISA. There were no significant increases in the expression of IL‐1β, IL‐8, IP‐10, SDF‐1, RANTES, Fractalkine, CX3CR‐1, CCR2, CCR5, CCR11, TNF‐α, GM‐CSF and TGF‐β using RT–PCR. There were no significantly increased levels of IL‐5, IL‐10, TNF‐α, TGF‐β, GROα, MIP‐1β and MCP‐1 found by ELISA in this study. Our results show that increased expression and secretion of IL‐6 in B19 NS1 transfected epithelial cells may play a role in the pathogenesis of autoimmune diseases.

Presentation of autoantibody to proliferating cell nuclear antigen in patients with chronic hepatitis B and C virus infection.

OBJECTIVES To study the association of antibodies to proliferating cell nuclear antigen (PCNA) in patients with chronic hepatitis B (HBV) and C (HCV) virus infection. METHODS Sera from 243 patients with chronic HBV infection; 379 patients with chronic HCV infection; 80 patients with systemic lupus erythematosus (SLE); 28 patients with rheumatoid arthritis; 15 patients with Sjogren’s syndrome; eight with polymyositis; eight with primary biliary cirrhosis; and 33 healthy control subjects were tested for the presentation of anti-PCNA antibodies by enzyme linked immunosorbent assay (ELISA) and immunoblotting using recombinant PCNA as antigen. The distribution of immunoglobulin isotypes of anti-PCNA antibody was measured by ELISA assay. RESULTS By ELISA, anti-PCNA antibodies were detected in 30 (12.3%) patients with chronic HBV infection, 71 (18.7%) patients with chronic HCV infection, and five (6.3%) patients with SLE. The inhibition of binding with these sera by purified PCNA was shown to exceed 71%. By immunoblotting, the frequency of anti-PCNA in patients with chronic HBV and HCV infection was 17 of 243 (7%) and 41 of 379 (11%), respectively. Absorption studies on indirect immunofluorescence showed the typical nuclear speckled staining pattern by anti-PCNA sera was abolished by preincubation of sera with PCNA. Anti-PCNA antibody was not detected in sera from patients with autoimmune diseases except SLE. Anti-PCNA antibodies in patients with chronic HBV and HCV infection were predominantly IgG. CONCLUSION These data suggest that anti-PCNA antibody are also present in patients with chronic HBV and HCV infection. Anti-PCNA antibody may not be specific for SLE.

Anti-CENP-H antibodies in patients with Sjogren's syndrome.

Anti-centromere antibody (ACA) has been reported to be associated with Sjogren’s syndrome (SS) and the clinical significance of anti-CENP-H antibody remains unknown. To determine the clinical significance of anti-CENP-H and anti-centromere antibodies in primary SS, sera from 62 patients with primary SS and 40 normal controls were examined for anti-SS-A/SS-B antibodies, ACA and anti-CENP-H antibodies, by enzyme-linked immunosorbent assay and indirect immunofluorescence (IIF), respectively. Of the 62 serum samples with primary SS, 17 were positive with ACA and anti-CENP-H antibodies. Sera from SS patients with anti-CENP-H and ACA antibodies do not contain anti-SS-A/Ro and/or anti-SS-B/La antibodies. No anti-CENP-H antibody was found in sera of normal controls. An increased frequency of ACA and anti-CENP-H antibodies was found for the first time in patients with SS. Anti-CENP-H antibodies and anti-SS-A/Ro or anti-SS-B/La antibodies are present mutually exclusive. Patients with anti-CENP-H antibodies had a lower frequency of rheumatoid factor (RF). SS can be subdivided serologically into two groups; group one with anti-SS-A/Ro and/or anti-SS-B/La antibody, group two with ACA and/or anti-CENP-H antibodies. We recommend that ACA or anti-CENP-H antibodies should be considered as one of the serological markers for SS.

Treatment with taurine attenuates hepatic apoptosis in NZB/W F1 mice fed with a high-cholesterol diet

Cholesterol-rich diets are known to cause hepatic apoptosis, which has been associated with the pathogenesis of systemic lupus erythematosus (SLE). However, the mechanisms and treatments for hepatic apoptosis in SLE are poorly understood. To clarify the effects of taurine on hepatic apoptosis in SLE, NZB/W F1 mice received control, cholesterol, and cholesterol/taurine diets. Significant reductions of caspase-3 activity, TUNEL-positive cells, and Fas- and mitochondrial- dependent apoptosis were detected in liver from the cholesterol/taurine group as compared to the cholesterol group. Moreover, significant increases of phosphorylated AKT, NF-kappaB (p65), and ERK1/2 proteins were detected in liver from the cholesterol/taurine group as compared to the cholesterol group. In contrast, a significant reduction of phosphorylated p38 protein was observed in the cholesterol/taurine group. These experimental results demonstrated positive effects of taurine against hepatic apoptosis in NZB/W F1 mice fed a high-cholesterol diet and suggested the therapeutic potential of taurine in SLE.

Beneficial effects of treatment with cystamine on brain in NZB/W F1 mice

The involvement of the central nervous system (CNS) or neuropsychosis has been reported in patients with systemic lupus erythematosus (SLE) and considered a major cause of long-standing functional impairment and mortality. However, little is known in the improvement of the brain abnormality in SLE. To investigate the effect and mechanism of cystamine on brain in SLE, NZB/W F1 mice were used as the animal model. Gel zymography, caspase-3 activity assay and Western blots were performed to elucidate the effect of cystamine. Significant reduction of matrix metalloproteinases (MMP)-9/MMP-2 ratio and urokinase-type plasminogen activator (uPA) expression was detected in brain of NZB/W F1 mice treated with cystamine as compared to control group. Significant increase of heat-shock protein (HSP)-70 and HSP27 was detected in brain of NZB/W F1 mice treated with cystamine as compared to control group. Additionally, significant reduction of mitochondrial dependent apoptosis was observed in brain of NZB/W F1 mice treated with cystamine as compared to control group by increasing BCL-2 and reducing caspase-9, Bad, and Apaf-1 expression. Moreover, increased phosphorylated p65 (NF-kappaB) protein was observed in brain of NZB/W F1 mice treated with cystamine as compared to control group. These experimental results firstly demonstrated the beneficial effects of cystamine on brain in NZB/W F1 mice and suggested the therapeutic potential in patients with neuropsychiatric SLE (NP-SLE).

Beneficial Effects of Ocimum gratissimum Aqueous Extract on Rats with CCl(4)-Induced Acute Liver Injury.

Ocimum gratissimum (OG) is known as a food spice and traditional herb, which has been recommended for the treatment of various diseases. To investigate the hepatoprotective effect of OG aqueous extract (OGAE), male Wistar rats challenged by carbon tetrachloride (CCl4) were used as the animal model of chronic hepatic injury. Significantly increased serum catalase and DPPH levels were detected in CCl4-administrated rats that were treated with OGAE or silymarin as compared to those rats that were treated with saline or CCl4. In contrast, significantly decreased stress proteins including HSP70 and iNOS were observed in livers of CCl4-administrated rats that were treated with OGAE or sylimarin as compared to those rats that were treated with saline or CCl4. Moreover, significant decreases of MMP-9/MMP-2 ratio, uPA, phosphorylated ERK (p-ERK) and NF-κB (p-P65) were detected in livers of CCl4-administrated rats that were treated with OGAE or sylimarin as compared to those rats that were treated with saline or CCl4. These findings imply that OGAE can efficiently inhibit CCl4-induced liver injuries in rats and may therefore be a potential food or herb for preventing liver injuries.