Advancement in the Development of Models for Hepatitis C Research

Hepatitis C virus (HCV) is a pandemic disease affecting an estimated 180 million individuals worldwide and infecting each year another ~3-4 million people making HCV a global public health issue. HCV is the main cause for chronic hepatitis, cirrhosis, and hepatocellular carcinoma. In the United States, HCV-related chronic liver disease is a leading cause of liver transplantation. Despite significant improvements in antiviral drugs, only ~50% of treated patients with HCV have viral clearance after treatment. Showing unique species specificity, HCV has a narrow range of potential hosts infecting only chimpanzees and humans. For decades, the chimpanzee model has been the only and instrumental primate for studying HCV infection; however, availability, economic, and ethical issues make the chimpanzee an unsuitable animal model today. Thus, significant research has been devoted to explore different models that are suitable in studying the biology of the virus and application in the clinical research for developing efficient and tolerable treatments for patients. This review focuses on experimental models that have been developed to date and their findings related to HCV

Pathogenic effect of interleukin-17A in induction of Sjogren's syndrome-like disease using adenovirus-mediated gene transfer

Introduction Sjögren's syndrome (SS) involves a chronic, progressive inflammation primarily of the salivary and lacrimal glands leading to decreased levels of saliva and tears resulting in dry mouth and dry eye diseases. Seminal findings regarding TH17 cell populations that secrete predominantly interleukin (IL)-17A have been shown to play an important role in an increasing number of autoimmune diseases, including SS. In the present study, we investigated the function of IL-17A on the development and onset of SS. Methods Adenovirus serotype 5 (Ad5) vectors expressing either IL-17A or LacZ were infused via retrograde cannulation into the salivary glands of C57BL/6J mice between 6 and 8 weeks of age or between 15 and 17 weeks of age. The mice were characterized for SS phenotypes. Results Disease profiling indicated that SS-non-susceptible C57BL/6J mice whose salivary glands received the Ad5-IL17A vector developed a SS-like disease profile, including the appearance of lymphocytic foci, increased cytokine levels, changes in antinuclear antibody profiles, and temporal loss of saliva flow. Conclusions Induction of SS pathology by IL-17A in SS-non-susceptible mice strongly suggests that IL-17A is an important inflammatory cytokine in salivary gland dysfunction. Thus, localized anti-IL17 therapy may be effective in preventing glandular dysfunction.National Institute of Dental and Craniofacial Research (U.S.) (PHS Grants K99DE018958)National Institute of Allergy and Infectious Diseases (U.S.) (R21AI081952)Sjogren's Syndrome FoundationUniversity of Florida. Center for Orphaned Autoimmune DisordersNational Institute of Dental and Craniofacial Research (U.S.) (Intramural research grant)National Institutes of Health (U.S.

Reduced IgG anti-small nuclear ribonucleoprotein autoantibody production in systemic lupus erythematosus patients with positive IgM anti-cytomegalovirus antibodies

INTRODUCTION: Systemic lupus erythematosus is characterized by production of autoantibodies to RNA or DNA-protein complexes such as small nuclear ribonucleoproteins (snRNPs). A role of Epstein-Barr virus in the pathogenesis has been suggested. Similar to Epstein-Barr virus, cytomegalovirus (CMV) infects the majority of individuals at a young age and establishes latency with a potential for reactivation. Homology of CMV glycoprotein B (UL55) with the U1snRNP-70 kDa protein (U1-70 k) has been described; however, the role of CMV infection in production of anti-snRNPs is controversial. We investigated the association of CMV serology and autoantibodies in systemic lupus erythematosus. METHODS: Sixty-one Mexican patients with systemic lupus erythematosus were tested for CMV and Epstein-Barr virus serology (viral capsid antigen, IgG, IgM) and autoantibodies by immunoprecipitation and ELISA (IgG and IgM class, U1RNP/Sm, U1-70 k, P peptide, rheumatoid factor, dsDNA, beta2-glycoprotein I). RESULTS: IgG anti-CMV and IgM anti-CMV were positive in 95% (58/61) and 33% (20/61), respectively, and two cases were negative for both. Clinical manifestation and autoantibodies in the IgM anti-CMV+ group (n = 20) versus the IgM anti-CMV(-)IgG+ (n = 39) group were compared. Most (19/20) of the IgM anti-CMV+ cases were IgG anti-CMV+, consistent with reactivation or reinfection. IgM anti-CMV was unrelated to rheumatoid factor or IgM class autoantibodies and none was positive for IgM anti-Epstein-Barr virus-viral capsid antigen, indicating that this is not simply due to false positive results caused by rheumatoid factor or nonspecific binding by certain IgM. The IgM anti-CMV+ group has significantly lower levels of IgG anti-U1RNP/Sm and IgG anti-U1-70 k (P = 0.0004 and P = 0.0046, respectively). This finding was also confirmed by immunoprecipitation. Among the IgM anti-CMV(-) subset, anti-Su was associated with anti-U1RNP and anti-Ro (P < 0.05). High levels of IgG anti-CMV were associated with production of lupus-related autoantibodies to RNA or DNA-protein complex (P = 0.0077). CONCLUSIONS : Our findings suggest a potential role of CMV in regulation of autoantibodies to snRNPs and may provide a unique insight to understand the pathogenesis

Induction of Cytoplasmic Rods and Rings Structures by Inhibition of the CTP and GTP Synthetic Pathway in Mammalian Cells

Background: Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined. Methodology/Principal Findings: Distinct cytoplasmic rods (,3–10 mm in length) and rings (,2–5 mm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in.95 % of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (.95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin

The Current Concept of TH17 Cells and Their Expanding Role in Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with a multifaceted range of symptoms affecting almost every organ system. The prototypical pathology of SLE involves the production of antinuclear antibodies and the deposition of immune complexes in basement membranes throughout the body where they induce inflammatory responses. The genetic and environmental etiologies of this process are being intensively sought, and recently, TH17 cells have been implicated in the pathogenesis of SLE. TH17 cells are CD4+ memory T cells that behave as both helper and effector cell populations functioning through their signature IL-17 cytokines. Their differentiation is distinct to either the TH1 or TH2 cell lineage, but strongly influences development of adaptive responses, including autoimmunity. This paper details the biological functions and regulation of TH17 cells, followed by an update of their expanding role in SLE.National Institute of Dental and Craniofacial Research (U.S.) (PHS Grants K99DE018958)National Institute of Allergy and Infectious Diseases (U.S.) (R21AI081952)National Institute of Allergy and Infectious Diseases (U.S.) (R01AI45050)Sjogren's Syndrome FoundationUniversity of Florida. Center for Orphaned Autoimmune Disorder

IL-22 regulation of functional gene expression in salivary gland cells

ABSTRACT: TH17 cells and their associated signature cytokines, IL-17 and IL-22, are highly elevated in primary Sjögren's syndrome (pSjS). The levels of IL-22 present in sera showed significant correlations with many disease parameters, specifically hyposalivation, anti-SSB, anti-SSA/SSB, hypergammaglobulinemia and rheumatoid factor. The present study aims to examine the biological function of IL-22 on human salivary glands. To accomplish the goal, microarray analysis using the HumanHT-12 v4 Expression BeadChip was utilized to determine the biological function of IL-22. Differential expression analyses were conducted using the LIMMA package from the Bioconductor project. MTT assay, flow cytometry and Western blotting were used to identify the function of IL-22 on human salivary gland cells. Results indicate an extensive effect of IL-22 on many major molecular functions including activation of antimicrobial genes and downregulation of immune-associated pathways. Functional studies performed in-vitro using human salivary gland cells treated with IL-22 indicated a direct effect of IL-22 on cell cycling, specifically reducing cellular proliferation at the G2-M phase by activation of STAT3. These results suggest the important role of IL-22 in the salivary gland function. The present study suggests that IL-22 might be involved in regulating inflammation and controlling the cell proliferation in SjS. Keywords: IL-22, Cytokine, Sjogren's syndrome, Gene expression, Microarra

The distribution of cytoplasmic rods and rings was independent of the Golgi complex and centrosomes, and these structures were not enriched in tubulin or vimentin.

<p>(A) Merged image of HEp-2 co-stained with human anti-RR prototype serum 604/Alexa 488 goat anti-human Ig (green) and rabbit anti-giantin (Golgi marker)/Alexa 568 goat anti-rabbit Ig (red). Rods are often presented adjacent (short arrows) or perpendicular (long arrows) to the nucleus while rings (arrowheads) are found either 1 or 2 to a cell. M, mitotic cell. HEp-2 cells were also co-stained with serum 604/Alexa 488 goat anti-human Ig (green, B,E,H) and different cytoplasmic markers using mouse anti-tubulin (C), anti-vimentin (F), anti-pericentrin (I), followed by Alexa 568 goat anti-mouse Ig (red). Nuclei were counterstained with DAPI (blue). Bar, 10 µm.</p

Concentration-dependent induction of RR in HEp-2 cells using different CTPS1 and IMPDH2 inhibitors for 24 h.

1<p>Ribavirin induced RR in nearly all cells regardless of the tested concentration (0.2 µM–2 mM). There is no statistical significance (ns) between concentrations after Bonferroni's adjustment except with untreated control (0 µM; p>0.005);</p>2<p>DON induced RR in most cells at concentrations higher than 0.5 mM (p<0.005, 0.5 mM or higher vs any lower concentration);</p>3<p>Acivicin started to induce RR at 15.6 µM and concentrations higher than 31.3 µM, nearly all cells had RR (p<0.005 vs lower concentration) and no difference between concentrations was observed;</p>4<p>ns between Ribavirin, DON, or Acivicin;</p>5<p>Ribavirin vs DON, p<0.0001, Ribavirin vs Acivicin, ns, DON vs Acivicin, p<0.0001;</p>6<p>Ribavirin vs DON, p<0.0001, Ribavirin vs Acivicin, p<0.0001, DON vs Acivicin, p = 0.024;</p>7<p>Ribavirin vs DON, p<0.0001, Ribavirin vs Acivicin, p<0.0001, DON vs Acivicin, ns.</p