Endocytosis of the Flaviviridae viruses, hepatitis C virus, GB virus C͞hepatitis G virus, and bovine viral diarrheal virus (BVDV) was shown to be mediated by low density lipoprotein (LDL) receptors on cultured cells by several lines of evidence: by the demonstration that endocytosis of these virus correlated with LDL receptor activity, by complete inhibition of detectable endocytosis by anti-LDL receptor antibody, by inhibition with anti-apolipoprotein E and -apolipoprotein B antibodies, by chemical methods abrogating lipoprotein͞LDL receptor interactions, and by inhibition with the endocytosis inhibitor phenylarsine oxide. Confirmatory evidence was provided by the lack of detectable LDL receptor on cells known to be resistant to BVDV infection. Endocytosis via the LDL receptor was shown to be mediated by complexing of the virus to very low density lipoprotein or LDL but not high density lipoprotein. Studies using LDL receptor-deficient cells or a cytolytic BVDV system indicated that the LDL receptor may be the main but not exclusive means of cell entry of these viruses. Studies on other types of viruses indicated that this mechanism may not be exclusive to Flaviviridae but may be used by viruses that associate with lipoprotein in the blood. These findings provide evidence that the family of LDL receptors may serve as viral receptors. H epatitis C virus (HCV), the principal viral cause of chronic hepatitis, is not readily replicated in cell culture systems, and, as yet, no information on cell receptors for the virus is available. However, several observations from studies on the role of HCV in mixed cryoglobulinemia (1-3) have provided some insights to HCV entry into cells.Mixed cryoglobulinemia is a systemic vasculitis associated with cold-precipitable immunoglobulins in the blood. During the past 5 years, a strong association of HCV infection with mixed cryoglobulins has been established (4), and the specific concentration of HCV in type II mixed cryoglobulins that consist of polyclonal IgG and monoclonal IgM has been demonstrated (1). It also was shown that very low density lipoprotein (VLDL) is selectively associated with HCV in type II cryoglobulins (2). In studies on the cutaneous vasculitic lesions in type II cryoglobulinemia using in situ hybridization (ISH), the HCV RNA virion form (positive strand) but not the putative replicative form (negative strand) of the virus was detected in keratinocytes in lesions but not normal skin of the same patients (3). Furthermore, it was demonstrated that LDL receptors were upregulated on keratinocytes in cutaneous vasculitis lesions compared with normal skin (3). These observations and the finding that anti-␤ lipoprotein precipitates HCV from infected serum (5) suggested that the low density lipoprotein (LDL) receptor also may be a receptor for HCV complexed to VLDL or LDL. This hypothesis led to this study to determine whether the LDL receptor is also a receptor for HCV and other members of the family of viruses Flaviviridae. Materials and Methods

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Type I1 mixed cryoglobulinemia is characterized by symptoms of systemic vasculitis and hepatic involvement. The cryoglobulins consist of polyclonal IgG complexed with monoclonal IgM rheumatoid factors (RF) that predominantly bear the WA crossidiotype (XId). Because of the high prevalence of hepatocellular abnormalities associated with the disease, hepatotropic viruses have been suspected as causative agents. Although hepatitis B virus was implicated in the pathogenesis of essential mixed cryoglobulinemia in early studies, evidence for ongoing hepatitis B infection in these patients was rarely found.Recently, an association of hepatitis C virus infection with type I1 mixed cryoglobulinemia has been established. Hepatitis C virus infection occurs in type I1 mixed cryoglobulinemia patients most commonly in association with WA monoclonal RF (mRF). This association raises the hypothesis that WA mRF is an antibody that cross-reacts with hepatitis C virus and that the virus is the causative agent of type I1 mixed cryoglobulinemia. The possibility that hepatitis C virus and WA RF play a role in mixed cryoglobu-

The controversial question of the extent of hepatocyte infection in chronic hepatitis C was re-examined in both chimpanzees and humans using a newly modified in situ hybridization (ISH) method for detecting hepatitis C virus (HCV) RNA. The specificity of the methodology for distinguishing positive-and negative-strand synthetic HCV RNA was at least six magnitudes greater than the reversetranscription polymerase chain reaction (RT-PCR) assay for HCV. The sensitivity of the methodology as determined by cell culture assay was l4 ؎ 2 genomic equivalents (gE) of HCV positive strand per cell, which was three magnitudes less sensitive than RT-PCR quantitation of HCV. In contrast to previous studies in both humans and chimpanzees with chronic hepatitis C, a high percentage of hepatocytes positive for both positive-and negative-strand HCV RNA was found in most specimens studied. In humans, the extent of hepatocyte infection varied with histological activity index (HAI). In the two chimpanzees studied, the liver biopsies showed minimal histological disease activity, but high percentages of hepatocytes were HCV-positive by ISH that correlated with hepatocyte ultrastructural changes associated with HCV infection. Hepatocyte infection was confirmed by RNA extraction and RT-PCR techniques for detecting HCV RNA that minimize the false detection of negative strands. In both human and chimpanzee liver biopsies showing minimal HAI, the hepatocyte concentration of HCV was estimated to be very low. These findings suggested the hypothesis that persistent infection in the liver may be caused in part by low-level HCV replication. The theoretical and clinical implications of these findings are discussed. (HEPATOLOGY 1998;28:573-584.)Extensive in situ hybridization (ISH) and immunohistochemical studies of chronic hepatitis C in humans have found that only small percentages of hepatocytes are infected. A study of two chimpanzees in which chronic hepatitis C developed after acute infection (Dr. Robert Purcell, personal communication) demonstrated, using ISH, that RNA was present in most hepatocytes within 2 days of infection but was no longer detectable at 20 weeks. 1 Studies of chronic hepatitis C in humans using ISH for detection of hepatitis C virus (HCV) RNA or immunohistochemical techniques for detection of HCV antigens in the liver produced some conflicting results with both techniques, but the findings in most of the studies using these two different methodologies were similar, with approximately 50% and 70% of liver specimens positive and less than 5% and 10% of hepatocytes positive by ISH and antigen detection, respectively. 2 Recent electron-microscopic studies for HCV were also consistent with previous in situ studies; HCV particles were rarely found in acute hepatitis C in chimpanzees and not at all in chronic hepatitis C in humans. 3 The finding of only small percentages of hepatocytes positive for HCV in chronic hepatitis C is puzzling because the most prominent pathological effect of HCV infection is hepatitis. In preliminar...

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Objective. To investigate the role of hepatitis C virus (HCV) in the pathogenesis of the cutaneous vasculitis in patients with type I1 cryoglobulinemia.Methods. Using in situ hybridization detection of HCV, we studied 6 test patients and various control subjects. Serum HCV was quantitated, cryoglobulins were analyzed by column chromatography at 37"C, and lowdensity lipoprotein (LDL) receptors on keratinocytes were detected using LDL labeled with fluorescent dye.Results. In the cutaneous vasculitic lesions from test patients, but not control subjects, the HCV virion was found in association with IgM and IgG. HCV alone was detected in some vessel walls, and in skin and ductal epithelium and vascular endothelium in inflamed, but not normal, skin. Cryoglobulins showed HCV, monomeric IgM, and monomeric IgG, with little or no immune complexes. The extent of the lesions correlated with levels of viremia. Up-regulation of LDL receptors on keratinocytes was detected in inflamed, but not normal, skin.Conclusion. HCV was present in the cutaneous vasculitic lesions, most likely in complexes with IgM and IgG formed in situ. These findings and the correlation of the severity of the rash with the level of viremia suggest that HCV plays a major role in the pathogenesis of cutaneous vasculitis in these patients and strength- The hallmark of type TI cryoglobulinemia is palpable purpura, a cutaneous vasculitis thought to be caused by the deposition of the components of cryoglobulins, polyclonal IgG, and monoclonal rheumatoid factor (RF) in the vessel walls (1). A strong association of hepatitis C virus (HCV) infection and type I1 cryoglobulinemia has been established (2,3), and the virus is specifically concentrated in the cryoglobulins (4). These findings suggest that HCV is the etiologic agent for the disease and that the virus may be involved in the pathogenesis of the systemic vasculitis that occurs in these patients.HCV is a single-stranded RNA virus, and both the positive strand, the putative virion RNA, and the negative strand, the putative replicative RNA, have been detected in hepatocytes of infected patients by polymerase chain reaction (PCR) assays ( 5 ) and by in situ hybridization and in situ PCR (6). HCV RNA has not been reported to be localized in cutaneous vasculitic lesions, but HCV has been detected in RNA extracted from these lesions using a PCR assay (7). However, because of the exquisite sensitivity of the PCR assay, the extraction technique does not distinguish between HCV RNA in the lesion and HCV RNA that may be present in small amounts of residual serum in unaffected blood vessels.We investigated the role of HCV in the pathogenesis of the cutaneous vasculitis in type I1 cryoglobulinemia by examining biopsy specimens of the palpable purpuric lesions from patients with type I1

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