Search

Background & Aims: CCL25 mediates the homeostatic recruitment of CCR9-expressing lymphocytes to the small intestine, but the function of this chemokine/receptor pair during chronic small intestinal inflammation has yet to be determined. Furthermore, although clinical trials to evaluate the efficacy of targeting the CCL25/CCR9 axis for the treatment of Crohn's disease are being conducted, preclinical data in animal models of IBD are lacking. Methods: In the current studies, we investigated the expression of CCL25 and CCR9 as a function of disease progression in a spontaneous murine model of chronic ileitis (SAMP1/YitFc) using flow cytometry, real-time reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. In addition, we assessed the functional role of the axis in the overall disease process through therapeutic studies that target the chemokine or the receptor during early and late disease. Results: The percentage of CCR9-expressing lymphocytes increased during early disease, accompanied by the appearance of a population of CCR9 high lymphocytes, predominantly within CD8 + T cells. Yet different from patients with primary sclerosing cholangitis, the expression of CCL25 remained restricted to the small intestine, even in mice with inflammation of the biliary tree. Neutralization of the receptor or the chemokine attenuated early disease but showed no therapeutic efficacy during the later stages, when overall CCR9 expression decreased and the CCR9 high population was absent. Conclusions: Our studies show that the role of this chemokine axis is not limited to homeostatic recruitment, as previously believed. However, these molecules appear to play their most crucial role during the early stages of chronic murine ileitis.

The cellular immune response plays a central role in virus clearance and pathogenesis of liver disease in hepatitis C. The study of hepatitis C virus (HCV)-specific immune responses is limited by currently available cell-culture systems. Here, the establishment and characterization of stable human HLA-A2-positive B-lymphoblastoid×T hybrid cell lines constitutively expressing either the NS3–4A complex or the entire HCV polyprotein are reported. These cell lines, termed T1/NS3-4A and T1/HCVcon, respectively, were maintained in continuous culture for more than 1 year with stable characteristics. HCV structural and non-structural proteins were processed accurately, indicating that the cellular and viral proteolytic machineries are functional in these cell lines. Viral proteins were found in the cytoplasm in dot-like structures when expressed in the context of the HCV polyprotein or in a perinuclear fringe when the NS3–4A complex was expressed alone. T1/NS3-4A and T1/HCVcon cells were lysed efficiently by HCV-specific cytotoxic T lymphocytes from patients with hepatitis C and from human HLA-A2.1 transgenic mice immunized with a liposomal HCV vaccine, indicating that viral proteins are processed endogenously and presented efficiently via the major histocompatibility complex class I pathway. In conclusion, these cell lines represent a unique tool to study the cellular immune response, as well as to evaluate novel vaccine and immunotherapeutic strategies against HCV.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) belongs to a class of membrane proteins termed tail-anchored proteins. Here, we show that the HCV RdRp C-terminal membrane insertion sequence traverses the phospholipid bilayer as a transmembrane segment. Moreover, the HCV RdRp was found to be retained in the endoplasmic reticulum (ER) or an ER-derived modified compartment both following transient transfection and in the context of a subgenomic replicon. An absolutely conserved GVG motif was not essential for membrane insertion but possibly provides a docking site for transmembrane protein-protein interactions. These findings have important implications for the functional architecture of the HCV replication complex.The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) belongs to a class of membrane proteins termed tail-anchored proteins. Here, we show that the HCV RdRp C-terminal membrane insertion sequence traverses the phospholipid bilayer as a transmembrane segment. Moreover, the HCV RdRp was found to be retained in the endoplasmic reticulum (ER) or an ER-derived modified compartment both following transient transfection and in the context of a subgenomic replicon. An absolutely conserved GVG motif was not essential for membrane insertion but possibly provides a docking site for transmembrane protein-protein interactions. These findings have important implications for the functional architecture of the HCV replication complex.