Tzy-Yen Chen

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.

Autophagy Inhibition Enhances Apoptosis Induced by Dioscin in Huh7 Cells

Extensive research results support the application of herbal medicine or natural food as an augment during therapy for various cancers. However, the effect of dioscin on tumor cells autophagy has not been clearly clarified. In this study, the unique effects of dioscin on autophagy of hepatoma cells were investigated. Results found that dioscin induced caspase-3- and -9-dependent cell apoptosis in a dose-dependent manner. Moreover, inhibition of ERK1/2 phosphorylation significantly abolished the dioscin-induced apoptosis. In addition, dioscin triggered cell autophagy in early stages. With autophagy inhibitors to hinder the autophagy process, dioscin-induced cell apoptosis was significantly enhanced. An inhibition of caspase activation did not affect the dioscin-induced LC3-II protein expression. Based on the results, we believed that while apoptosis was blocked, dioscin-induced autophagy process also diminished in Huh7 cells. In conclusion, this study indicates that dioscin causes autophagy in Huh7 cells and suggests that dioscin has a cytoprotective effect.

Human parvovirus B19 infection in patients with chronic hepatitis B or hepatitis C infection.

Background and Aim:  Parvovirus B19 has been reported to be detected in the sera of patients with acute or chronic hepatitis. The prevalence and clinical significance of B19 DNA in serum samples from patients with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection were investigated.

Anti-PCNA autoantibodies preferentially recognize C-terminal of PCNA in patients with chronic hepatitis B virus infection

We previously reported anti‐PCNA autoantibodies in sera from patients with chronic HBV and HCV infection. To analyse the antigenic regions on proliferating cell nuclear antigen (PCNA) that confer autoantibody binding in patients with chronic hepatitis B (HBV) and C (HCV) infection, eight constructs including one wild type PCNA, one mutant type Y114A_PCNA and six C‐ or N‐terminal PCNA truncations were generated. Sera from 185 patients with systemic lupus erythematosus (SLE), 178 with chronic HBV and 163 with chronic HCV infection, and 68 healthy individuals were examined for the presentation of anti‐PCNA antibodies by enzyme linked immunosorbent assay (ELISA). By ELISA, anti‐PCNA positive sera from patients with SLE, chronic HBV and HCV infection preferentially recognized the wild type PCNA more than the mutant type Y114A_PCNA (P < 0·05). The inhibition of binding by purified full‐length rPCNA proteins with anti‐PCNA positive sera was shown to exceed 70%. The inhibition of binding by purified truncated rPCNA proteins with sera from patients with chronic HBV and HCV infection and SLE was shown to confer dominant binding in TL2 and TL3. Moreover, the higher frequency of inhibition by using TL3 was found in patients with chronic HBV infection. These data indicate that anti‐PCNA autoantibodies preferentially recognize C‐terminal of PCNA in patients with chronic HBV infection and may also provide advanced understanding between viral infection and autoimmunity for further study.

CD44 Gene Polymorphisms on Hepatocellular Carcinoma Susceptibility and Clinicopathologic Features

Hepatocellular carcinoma (HCC) is the second leading cause of cancer deaths in Taiwan. CD44, one of the well-known tumor markers, plays an essential role in tumor cell differentiation, invasion, and metastasis. We investigated the CD44 single-nucleotide polymorphisms (SNPs) with environmental risk factors related to HCC susceptibility and clinicopathological characteristics. Six SNPs of CD44 were analyzed using a real-time polymerase chain reaction (PCR) in 203 patients with HCC and in 561 cancer-free controls. We determined that the individuals carrying at least one G allele at CD44 rs187115 has higher risk of developing HCC than did wild-type (AA) carriers. We further observed that the CD44 rs187115 polymorphisms with at least one G allele had a higher frequency of distribution in nonsmoking stage III/IV HCC patients, compared with wild-type carriers. Our results suggested that patients with CD44 rs187115 variant genotypes (AG+GG) were associated with a higher risk of HCC development and that these patients might possess chemoresistance, causing more likely progression to late-stage HCC than wild-type carriers without the overexpression of CD44 induced by heavy smoking. CD44 rs187115 might be involved in CD44 isoform expression of p53 stress response in HCC and provide a marker for predicting worst-case prognosis of HCC.

Human parvovirus B19 VP1u Protein as inflammatory mediators induces liver injury in naïve mice

Human parvovirus B19 (B19V) is a human pathogen known to be associated with many non-erythroid diseases, including hepatitis. Although B19V VP1-unique region (B19-VP1u) has crucial roles in the pathogenesis of B19V infection, the influence of B19-VP1u proteins on hepatic injury is still obscure. This study investigated the effect and possible inflammatory signaling of B19-VP1u in livers from BALB/c mice that were subcutaneously inoculated with VP1u-expressing COS-7 cells. The in vivo effects of B19-VP1u were analyzed by using live animal imaging system (IVIS), Haematoxylin-Eosin staining, gel zymography, and immunoblotting after inoculation. Markedly hepatocyte disarray and lymphocyte infiltration, enhanced matrix metalloproteinase (MMP)-9 activity and increased phosphorylation of p38, ERK, IKK-α, IκB and NF-κB (p-p65) proteins were observed in livers from BALB/c mice receiving COS-7 cells expressing B19-VP1u as well as the significantly increased CRP, IL-1β and IL-6. Notably, IFN-γ and phosphorylated STAT1, but not STAT3, were also significantly increased in the livers of BALB/c mice that were subcutaneously inoculated with VP1u-expressing COS-7 cells. These findings revealed the effects of B19-VP1u on liver injury and suggested that B19-VP1u may have a role as mediators of inflammation in B19V infection.

Cystamine attenuates lupus-associated apoptosis of ventricular tissue by suppressing both intrinsic and extrinsic pathways

Cystamine, a disulphide metabolite, has been demonstrated to ameliorate various lupus‐associated tissue damages by animal models. However, effects of cystamine on apoptosis of cardiac tissue, a main cardiac damage attributing to lupus, are less obvious. Therefore, we aimed to investigate whether or not cystamine possesses anti‐apoptotic effects with emphasis on LV tissue of lupus‐prone mice NZB/W‐F1. Cystamine treatment was performed by daily intraperitoneal administration. Morphology and apoptotic status of ventricular tissues in the treated mice were assessed by microscopy and TUNEL assay, respectively. Levels of apoptotic biomarkers were determined using immunoblot. Our results revealed that cystamine significantly attenuated the apoptosis of LV tissues in NZB/W‐F1 mice, whereas the morphology of the tissues was slightly altered. In addition, cystamine reduced level of Fas and inhibited activation of caspase‐8. Cystamine also increased level of Bcl‐2 and phosphorylation of Bad, and decreased level of Bad and truncated Bid (tBid). Moreover, level of cytosolic cytochrome c and Apaf‐1, and activation of caspase‐9 and caspase‐3 were suppressed in response to cystamine treatment. In Balb/c mice, as normal control mice, changes in cell morphology and levels of the tested apoptotic components were found insignificant in the LV tissues. These findings indicate that cystamine treatment attenuates apoptosis of LV tissues of NZB/W‐F1 mice through suppressing both intrinsic and extrinsic apoptotic pathways. Therefore, cystamine is considered beneficial to alleviating lupus‐associated cardiac damages.

The Involvement of Mitochondrial-related Caspase Pathway in HCV NS5A-induced Apoptosis of Huh-7 Cells

Chronic HCV infection may lead to hepatic fibrosis and the precise mechanisms remain unclear. In this study, Huh-7 cells were transiently transfected with NS5A and subjected to MTT assay, DNA fragmentation assay and western blotting to see the impact of NS5A protein on apoptosis. While cell cycle was not affected, NS5A may inhibit cell proliferation by inducing apoptosis since pro-caspases 3, 8 & 9 were cleaved result in the presence of active forms in a time-dependent fashion. These findings suggested that NS5A-induced apoptosis is caspase-dependent. Furthermore, the cytosolic level of cytochrome c was increased together with a gradually down-regulated Bcl-2 and up-regulated Bax protein expression. The persistent reduction of Bid protein and the gradual increase of tide protein also indicated that a time-dependent increased turn-over of Bid protein into tide. Taken together, our data suggested that HCV NS5A may induce apoptosis through a mitochondrial- related caspase pathway.

Corrigendum to “Autophagy Inhibition Enhances Apoptosis Induced by Dioscin in Huh7 Cells”

[This corrects the article DOI: 10.1155/2012/134512.].

A Laboratory-Based Antibody Array for Profiling Cytokine Expression

Purpose: Despite the expansion in proteomics research, the instability and complexity of proteins limit the feasibility of adopting antibody arrays in laboratory studies. We developed a simple and rapid antibody array-based method to characterize cytokine expression and evaluated its validity and accuracy. Method: We spotted monoclonal antibodies against various cytokines onto a nitrocellulose slide (FAST(superscript TM) Slides) in tetrad with a microarray spotting system (SpotArray(superscript TM) 24) and stored the slides at -20°C until use, In addition, we subjected cells to lipopolysaccharide (LPS) and shear stress treatment to alter the cytokine secretion profiles and validated the antibody array method using reverse transcription-polymerase chain reaction (RT-PCR). Results: The sensitivity of the antibody array test was higher than 100 pg/ml. Variations in cytokine secretion due to LPS and shear stress-treated Raw 264.7 cells were detected by the array. The cytokine mRNA levels measured by the array were confirmed by RT-PCR analysis. The procedures took approximately 90 minutes. The antibody array reproduced similar results after being stored at -20°C for 6 months. Conclusion: The laboratory-based antibody array is efficient, accurate, and feasible for profiling cytokine expression.