Effector-memory T cells are strategically placed to epithelial tissues to provide frontline immune protection against pathogens. Their detrimental effects, however, have been rarely examined because of difficulty in sampling these T cells in pathological settings. Our previous studies suggested persistence of a similar subset of intraepidermal CD8(+) T cells at high frequencies in the lesions of fixed drug eruption, a localized variant of drug-induced dermatoses. In situ activation of this subset resulting in localized epidermal injury can be traced in the lesions after antigen challenge by paired immunohistochemical staining, reverse transcriptase-polymerase chain reaction in situ, and flow cytometry of dispersed cells. Here we show that effector-memory T cells were greatly enriched in these intraepidermal CD8(+) T cells, but not dermal and circulating counterparts, and that they constitutively express an early activation marker CD69 even before challenge. Surprisingly, a large proportion of these T cells expressed immediate effector function as evidenced by the rapid production of high levels of interferon-gamma in situ with much faster kinetics than their counterparts at the mRNA and protein levels after challenge. This was followed by localized epidermal injury. The intracellular cytokine assay ex vivo shows that the great majority of these dispersed T cells produce interferon-gamma. This study provides the first in situ description of the detrimental effects specifically mediated by effector-memory T cells residing at the effector site of immunopathology.

Hepatitis C virus (HCV) causes persistent infection inhepatocytes, and this infection is, in turn, strongly associated with the development of hepatocellular carcinoma. To clarify the mechanisms underlying these effects, we established a Cre/loxP conditional expression system for the precisely self-trimmed HCV genome in human liver cells. Passage of hepatocytes expressing replicable full-length HCV (HCR6-Rz) RNA caused upregulation of anchorage-independent growth after 44 days. In contrast, hepatocytes expressing HCV structural, nonstructural, or all viral proteins showed no significant changes after passage for 44 days. Only cells expressing HCR6-Rz passaged for 44 days displayed acceleration of CDK activity, hyperphosphorylation of Rb, and E2F activation. These results demonstrate that full genome HCV expression up-regulates the CDK-Rb-E2F pathway much more effectively than HCV proteins during passage.Hepatitis C virus (HCV) 1 causes the persistent infection chronic hepatitis in most infected patients. This disorder eventually progresses to cirrhosis and hepatocellular carcinoma (HCC). Numerous studies have provided evidence supporting a link between chronic HCV infection and HCC (1, 2). However, exactly how HCV infection could be directly involved in the development of HCC remains unclear because of the lack of an efficient in vitro infection system.HCV is a member of the Flaviviridae family and has a positive-strand RNA genome (ϳ9.6 kb). Viral proteins are synthesized as a single polyprotein, which is then cleaved into structural (core, E1, and E2) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins. In vitro expression of HCV core protein reportedly influences cell growth regulation, and the protein can interact with the cytoplasmic tail of a lymphotoxin-␤ receptor (3), the death domain of tumor necrosis factor receptor-1 (4), and NF-B (5), resulting in enhancement or inhibition of Fas-and tumor necrosis factor-␣-mediated apoptosis. HCV structural protein inhibits Fas-mediated apoptosis by inhibiting cytochrome c release from mitochondria in mice (6). Cellular transformation has been shown to be caused by HCV core protein in the presence of the ras gene (7,8) and by loss of function of LZIP (9) and the presence of STAT3 (signal transducer and activator of transcription-3) (10). NS3 (11) and NS4B (12) proteins reportedly display tumorigenicity in the presence of ras. These results indicate that expression of individual HCV proteins does not cause cellular transformation in vitro. HCV core protein transgenic mice reportedly show induced steatosis and, after 16 months of age, develop HCC (13). In contrast, HCV structural protein transgenic mice do not display neoplastic or cancerous lesions in the liver by 20 months of age (14). Moreover, conditional expression of an HCV structural protein region (nucleotides 294 -3435) causes hepatic injury in transgenic mice (15), but HCC is not observed by at least 16 months of age (data not shown). The frequency of HCC occurrence is reportedly higher in full-...

Persistent infection with hepatitis C virus (HCV) induces tumorigenicity in hepatocytes.To gain insight into the mechanisms underlying this process, we generated monoclonal antibodies on a genome-wide scale against an HCV-expressing human hepatoblastoma-derived cell line, RzM6-LC, showing augmented tumorigenicity. We identified 3␤-hydroxysterol ⌬24-reductase (DHCR24) from this screen and showed that its expression reflected tumorigenicity. HCV induced the DHCR24 overexpression in human hepatocytes. Ectopic or HCV-induced DHCR24 overexpression resulted in resistance to oxidative stress-induced apoptosis and suppressed p53 activity. DHCR24 overexpression in these cells paralleled the increased interaction between p53 and MDM2 (also known as HDM2), a p53-specific E3 ubiquitin ligase, in the cytoplasm. Persistent DHCR24 overexpression did not alter the phosphorylation status of p53 but resulted in decreased acetylation of p53 at lysine residues 373 and 382 in the nucleus after treatment with hydrogen peroxide. Taken together, these results suggest that DHCR24 is elevated in response to HCV infection and inhibits the p53 stress response by stimulating the accumulation of the MDM2-p53 complex in the cytoplasm and by inhibiting the acetylation of p53 in the nucleus. Hepatitis C virus (HCV)5 is composed of a single-stranded RNA genome of positive polarity (1). Translation of viral proteins is initiated from an internal ribosome entry site (2) and results in a single polypeptide that is subsequently cleaved by host and viral proteases to yield viable proteins (3). The HCV genome does not rely on canonical translation factors and can readily establish chronic infection without integrating into the host genome, resulting in hepatic steatosis and hepatocellular carcinoma (HCC) (4). More than 170 million people worldwide are infected with HCV (5); chronic HCV infection and aging are the major risk factors for HCC (6 -8). Liver cancer is the fifth most common cause of cancer mortality worldwide (9). The frequent inactivation of p53 in human HCC suggests that the loss of p53-dependent apoptosis may promote hepatocarcinogenesis (10). Chronic HCV infection results in chronic liver inflammation and induces endoplasmic reticulum stress and oxidative stress, which are thought to induce hepatocarcinogenesis (11, 12). The mechanistic details underlying HCC development are not fully understood. To gain insight into the molecular mechanisms underlying HCV-induced pathogenesis, we previously established RzM6 cells (13), a human hepatoblastoma (HepG2)-derived cell line in which expression of the full-length HCV genome is controlled by a Cre/loxP system. Expression of the HCV genome promoted anchorage-independent growth of RzM6 cells after 44 days of culture from the onset of HCV expression (RzM6-44d cells) but not in RzM6 cells after 0 days (RzM6-0d cells) (13). In the present study, we generated monoclonal antibodies against RzM6 cells cultured for longer than 44 days (RzM6-LC cells) and then screened the antibodies for their ability to...

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Hepatitis C virus (HCV) is the major causative agent of hepatocellular carcinoma. However, the precise mechanism underlying the carcinogenesis is yet to be elucidated. It has recently been reported that Syk, a non-receptor protein tyrosine kinase, functions as a potent tumour suppressor in human breast carcinoma. This study first examined the possible effect of HCV infection on expression of Syk in vivo. Immunohistochemical analysis revealed that endogenous Syk, which otherwise was expressed diffusely in the cytoplasm of normal hepatocytes, was localized near the cell membrane with a patchy pattern in HCV-infected hepatocytes. The possible interaction between HCV proteins and Syk in human hepatoma-derived Huh-7 cells was then examined. Immunoprecipitation analysis revealed that NS5A interacted strongly with Syk. Deletion-mutation analysis revealed that an N-terminal portion of NS5A (aa 1-175) was involved in the physical interaction with Syk. An in vitro kinase assay demonstrated that NS5A inhibited the enzymic activity of Syk and that, in addition to the N-terminal 175 residues, a central portion of NS5A (aa 237-302) was required for inhibition of Syk. Moreover, Syk-mediated phosphorylation of phospholipase C-c1 was downregulated by NS5A. An interaction of NS5A with Syk was also detected in Huh-7.5 cells harbouring an HCV RNA replicon or infected with HCV. In conclusion, these results demonstrated that NS5A interacts with Syk resulting in negative regulation of its kinase activity. The results indicate that NS5A may be involved in the carcinogenesis of hepatocytes through the suppression of Syk kinase activities. INTRODUCTIONHepatitis C virus (HCV) is the major aetiological agent of viral hepatitis worldwide after hepatitis A and B viruses (Choo et al., 1989), with about 170 million people being infected. The majority of HCV-infected individuals develop chronic infection, which may progress to liver cirrhosis and hepatocellular carcinoma (HCC). HCV is a member of the family Flaviviridae and its genome consists of a single-stranded, positive-sense RNA of approximately 9600 nt, which encodes a polyprotein precursor of about 3010 aa. Currently, clinical HCV isolates are classified into six genotypes and more than 60 subtypes (Doi et al., 1996;Mellor et al., 1995;Robertson et al., 1998). The polyprotein is cleaved by signal peptidase, signal peptide peptidase and two virally encoded proteases to generate at least ten mature proteins: core, envelope glycoprotein 1 (E1), E2, p7, non-structural protein 2 (NS2), NS3, NS4A, NS4B, NS5A and NS5B Reed & Rice, 2000).HCV NS5A is part of the replication complex that catalyses replication of the viral genome. NS5A takes two forms, p56 and p58, with different degrees of phosphorylation, which may play distinct roles in the virus replication cycle (Evans 3These authors contributed equally to this work. YXX(L/I)X 6-8 YXX(L/I)] in the cytoplasmic tail of the Fc receptor c-chain or B-cell receptor subunit Iga to be activated after the engagement of immune receptors (Kurosaki et al...