Myths, misconceptions and mixed cryoglobulinemia associated with HCV infection.

The delineation of the association of HCV infection with mixed cryoglobulinemia has provided new insight into the etiology and pathogenesis of this extrahepatic manifestation of the infection. Yet very little evidence has been obtained thus far on the specific roles of virus in production of the monoclonal rheumatoid factors responsible for classic type II cryoglobulins and the associated clinical manifestations. The problematic areas of investigation that have in some instances generated misconceptions due to lack of data are reviewed. These include the prevalence and heterogeneity of mixed cryoglobulins; clinical manifestations such as liver cirrhosis, membranoproliferative glomerulonephritis, autoimmunity, progression of cryoglobulinemia from type III to type II, development of B cell malignancies; determination of lineages based on immunoglobulin gene utilization; and the anti-viral treatment of patients with mixed cryoglobulinemia.

Use of whole blood specimens for routine clinical quantitation of hepatitis C virus RNA does not increase assay sensitivity.

The measurement of hepatitis C virus (HCV) RNA levels in the blood has, in the last few years, become a critical component in the therapy of patients with HCV infections. Initially, extraction methods for serum and plasma were used, but a newer method that uses Catrimox-14 as the extraction agent for whole blood has been reported. Because the whole blood extraction method may yield higher virus levels if significant levels of virus are present in the white blood cells (WBC), the method was evaluated for use in our clinical diagnostic laboratory despite its higher reagent costs and more time-consuming methodology. RNA was simultaneously extracted from 39 clinical samples by four different methods: Catrimox-14-Trizol extraction from whole blood, Trizol extraction from whole blood, Trizol extraction from serum, and a commercial serum extraction method, the EZNA total RNA kit. In addition, in an effort to quantitate the amount of HCV RNA virus in the WBC, Trizol extraction from isolated WBC was also performed. Quantitative results for samples from which RNA was extracted by all four methods were essentially the same; the Catrimox-14-Trizol method did not yield increased virus levels. Insignificant levels of virus were found in the WBC. The results did not demonstrate a clinical usefulness for the Catrimox-14-Trizol method.

Hepatitis C virus but not GB virus C/hepatitis G virus has a role in type II cryoglobulinemia.

OBJECTIVE: Hepatitis C virus (HCV) infection is associated with type II cryoglobulinemia. HCV is specifically concentrated in type II cryoglobulins and has been implicated in the cutaneous vasculitis associated with the disease. In contrast to HCV, a role for hepatitis G virus (HGV) in type II cryoglobulinemia has not been defined, although prevalences as high as 43% of HGV infections in type II cryoglobulinemia have also been reported. METHODS: We studied 34 patients with type II and 29 patients with type III cryoglobulinemia associated with HCV infection, 6 patients with essential mixed cryoglobulinemia (EMC; all with type II), 50 hospital control patients, and 125 normal individuals. Serum HCV and HGV RNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). In coinfected sera, HCV and HGV were quantitated by competitive RT-PCR assays. One coinfected patient was studied longitudinally for 6 years. RESULTS: Two (5.9%) of 34 patients with HCV-infected type II cryoglobulinemia, none of 29 patients with type III cryoglobulinemia, and none of 6 patients with EMC were positive for HGV RNA, for an overall prevalence of 3.0% in mixed cryoglobulinemia. None of the control populations were positive for HGV. No statistical difference was seen between the prevalence in patients with type II cryoglobulinemia and the other populations studied. In coinfected sera, HCV, but not HGV, was concentrated in cryoglobulins, and HCV, but not HGV, correlated with cryoglobulinemia in a longitudinal study. CONCLUSION: There is a low prevalence of coinfection with HGV in patients with mixed cryoglobulinemia and HCV infection in the United States. HCV is selectively precipitated by type II cryoglobulins in coinfected sera. HGV infection does not appear to have a role in mixed cryoglobulinemia.

Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor.

Endocytosis of the Flaviviridae viruses, hepatitis C virus, GB virus C/hepatitis 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 lipoprotein/LDL 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.

A competitive reverse transcription-polymerase chain reaction assay for quantitation of GB virus C/hepatitis G virus RNA that circumvents heteroduplex artifact.

The role of GB virus C (GBV-C)/hepatitis G virus (HGV) in hepatitis has been controversial. To investigate its possible pathogenicity and site(s) of replication, it is important to develop an accurate quantitative assay for both positive and negative strand GBV-C/HGV RNA. In this study, a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay for both positive and negative strand GBV-C/HGV RNA quantitation was developed. In developing the quantitative assay, heteroduplex formation was repeatedly observed. A heterologous competitor RNA with GBV-C/HGV primer-binding sequences was introduced, and heteroduplex artifact was circumvented successfully. Two-hundred thirty-seven serum specimens were screened by RT-PCR for GBV-C/HGV RNA. Two of the 62 patients infected with chronic hepatitis C virus (HCV) were found to be positive for GBV-C/HGV RNA. None of the 50 other patients with no evidence of HCV infection and none of the 125 normal individuals were positive for GBV-C/HGV RNA. The sensitivity of RT-PCR was 3000 gE/ml (30 gE in RT-PCR). Alternate methods for residual DNA removal and its detection in synthetic RNA were introduced. A RT control containing no primer before PCR is necessary to evaluate the trace amounts of template DNA remaining in synthesized RNA. The method will differentiate reliably between positive and negative strand RNAs up to a 10(4)-fold difference in titer. The positive and negative strand GBV-C/HGV RNAs were detected in one patient by RT-PCR and hybridization analysis, and the strand titer was determined by RT-PCR.

Detection of widespread hepatocyte infection in chronic hepatitis C.

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 reverse-transcription polymerase chain reaction (RT-PCR) assay for HCV. The sensitivity of the methodology as determined by cell culture assay was 14 +/- 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.

The epidemiology of migraine: a retrospective study in Italian emergency departments.

This study was conducted to measure the frequency of contact with emergency departments in Italy because of migraine, and to compare the initial diagnosi s of headache with the diagnosis after application of the International Headache Society (IHS) criteria. A retrospective observational method was used, consisting of an analysis of the records of patients admitted to nine Italian emergency departments during different 4-month periods in 1994. Comparison of the initial diagnosis with the diagnosis after application of the IHS diagnostic criteria was performed. More than 31 million emergency department contacts were reported in Italy during 1994. In the same year, 543 630 patients visited the nine emergency departments enrolled in the study, with 169 569 of these contacts occurring in the 4-month period analyzed in the study. We excluded from the analysis all cases of secondary headache fully recognized at the emergency department admission (ie, traumas, intracranial pathology, systemic diseases). The total number of patients included in our analysis was 1043 (0.6%). The 934 patients who could be fully evaluated were initially classified as having migraine; cluster headache; headache not otherwise specified; or diagnosed in the emergency department as suffering from headache, but reclassified by other departments as suffering from a different disease. After retrospective application of the IHS classification, the diagnostic distribution was modified, revealing that 18% of patients with migraine and 5% with cluster headaches had previously been classified as having headache not otherwise specified; a further 6% of cases with migraine and 0.4% of patients with cluster headache had previously been classified as having secondary headaches. The diagnosis of headache not otherwise specified was made with notable frequency, indicating the limits of emergency department logs and the difficulty in carrying out a retrospective analysis and reassessment of diagnosis. The majority (88%) of patients assessed had not taken drugs for headache in the 48 hours before the emergency department contact, suggesting that in Italy emergency departments are used instead of a visit to the general practitioner. Nonsteroidal anti-inflammatory drugs were the most frequently prescribed drugs in the emergency departments for this group of diagnoses. The research revealed, on the one hand, that headache is a numerically significant phenomenon in the emergency department setting and, on the other, the need to apply prospective designs to this kind of survey.

Predictors of long-term response to high-dose interferon therapy in type II cryoglobulinemia associated with hepatitis C virus infection.

We have prospectively studied patients with type II cryoglobulinemia since 1985 to assess the efficacy of treatment with interferon-alpha at cumulative doses ranging from 234 to 849 MU. In the present study we retrospectively evaluated in this cohort parameters associated with complete response to therapy in 31 consecutive patients with type II cryoglobulinemia associated with hepatitis C virus (HCV) infection. Prevalence of complete response of cryoglobulinemia (disappearance of symptoms and signs of vasculitis and decrease of cryocrit below 10% of the initial value) was 62%, with a median response duration of 33 months and a range of 3 to 100 months. Three patients were putatively cured, as they remained in complete remission for more than 5 years off therapy. Eighteen patients (58%) had liver disease evidenced by histopathology and/or raised transaminase levels. Prevalence of normalization of transaminase levels was 100%, with a median response duration of 36 months. Relapse of hypertransaminasemia occurred in 100% and 8% of patients receiving less than or greater than 621 MU, respectively. By logistic regression analysis, the only pretherapy parameter that associated significantly (P = .0393) with complete response of cryoglobulinemia was the solitary anti-C22 (HCV core) antibody pattern, which was observed in 29% of patients. Association with older age and low cryocrit approached statistical significance (P = . 06), while no significant correlations were found with serum IgM levels, duration of disease, HCV genotype, NS5a gene mutations, liver histology, HLA-DR phenotype, or WA cross-idiotype. Complete responses were also associated, on univariate statistical analysis, with low pretherapy HCV viremia. Responses were accompanied by decrease of viremia, of anti-HCV antibody levels and cryocrit. The usefulness of a high dose regimen is underscored by the higher rates of sustained responses of cryoglobulinemia and transaminase levels compared with previous studies.

Localization of hepatitis C virus in cutaneous vasculitic lesions in patients with type II cryoglobulinemia.

OBJECTIVE: To investigate the role of hepatitis C virus (HCV) in the pathogenesis of the cutaneous vasculitis in patients with type II 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 degrees C, and low-density 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 strengthens the rationale for antiviral drug therapy. The presence of HCV in keratinocytes and ductal epithelial and vascular endothelial cells may be the in vivo manifestation of endocytosis of HCV by the LDL receptors that has recently been demonstrated in vitro. The up-regulation of LDL receptors on keratinocytes in inflamed skin is consistent with this postulation.

A simplified method for Ca2+ flux measurement on isolated human B cells that uses flow cytometry.

A method for Ca2+ flux measurement on isolated human peripheral B cells that uses flow cytometry is described. B cells were isolated by anti-CD19 magnetic bead sorting, and Ca2+ flux was measured with the fluo-3 reagent on a standard single-laser flow cytometer. The response of B-cell stimulation by anti-immunoglobulin B (anti-IgM), anti-IgD, protein A, concanavalin A, and ionomycin was determined. Percentage of responder B cells, the level of Ca2+, and the time of peak stimulation were measured. Bound anti-CD19 monoclonal antibody coupled with small paramagnetic particles did not affect Ca2+ flux. All the isolated B cells responded maximally at 10s with stimulation by 8 microg of ionomycin. The average isolated preparation contains 70% IgM+ and 85% IgD+ cells, all of which showed peak stimulation with 10 microg of anti-IgM and anti-IgD per ml, respectively, at 30s. Only at high concentrations of 80 microg/ml, concanavalin A produced a slower response, peaking at 90 s after stimulation. Stimulation with 20 microg of protein A per ml resulted in Ca2+ flux in only 40 to 60% of cells that had a rapid response and maximal stimulation resembling the pattern of activation of ionomycin. B cells from three patients with mixed cryoglobulinemia with high concentrations of monoclonal rheumatoid factors showed stimulation with aggregated IgG, whereas those from healthy control subjects did not, demonstrating the applicability of the methodology to detection of specific antigen stimulation of B cells. This methodology may be useful in testing the functional capacity of B cells in a variety of diseases. The methodology may also prove useful in studying antigen-specific B-cell responses when they involve a significant percentage of B cells.

The etiology and pathophysiology of mixed cryoglobulinemia secondary to hepatitis C virus infection.

The strong association of HCV infection with MC-II and the selective concentration of the virus with the WA mRF in the cryoglobulins are compelling suggestions that the virus is directly involved in production of the mRF and the pathophysiology of MC-II. There is, however, only limited data on HCV involvement in both processes. In cutaneous vasculitis, which is the most prevalent clinical feature of the disease, there is evidence that complexes of HCV, mRF and IgG are formed in situ from components of the cryoglobulins that are present in the blood in a dissociated state. It is postulated that local factors, cooling and stasis predispose to formation of these lesions in the lower limbs. However, since cutaneous vasculitis does not correlate with cryoglobulin levels and may not be induced by cold challenge, other factors may be involved. In particular, the conditions which activate the vascular endothelial cells, leading to the leukocytoclastic vasculitis, require delineation. In contrast to cutaneous vasculitis, HCV RNA has not been prominently detected in immune complexes in MPGN lesions and has not been detected at all in the peripheral neuropathy lesions. These preliminary observations suggest that different pathophysiological processes are involved in for these lesions than in cutaneous vasculitis. From the correlation of remission of disease with decreased cryoglobulinemia and viremia in treated patients with MC-II, and from immunohistological data on the hepatitic lymphoid follicles in MC-II (see chapter 7), it appears that an antigen-driven benign proliferation of B cells is responsible for production off mRF and cryoglobulinemia. New findings have suggested that one mechanism for developing mixed cryoglobulinemia may be that HCV-VLDL complexes that contain apo E2 are poorly endocytosed by the LDLR, which may be a major route of entry of the virus to the cell; persistence of the complexes in the circulation may then stimulate mRF production. This new hypothesis is based only on initial in vitro observations and require independent confirmation and validation in vivo. From indirect clinical evidence it has also been postulated that mRF in some patients may limit the cytopathology in MC-II, resulting in a lower prevalence of cirrhosis in these patients. These findings suggested another hypothesis, which is that the mRF prevents spread of infection to hepatocytes and other permissive and nonpermissive cells by blocking endocytosis of HCV-VLDL complexes by the LDLR. Furthermore, data on the composition of cryoglobulins, the molecular composition of WA mRF and the characterization of monoclonal B cells in the liver of patients with MC-II (see chapter 7) suggest that a specific population of B cells may be involved in the host response to HCV infection. These are B cells that proliferate with little or no somatic mutations of the immunoglobulin genes, are self-replicating, are stimulated by self antigens in a T cell-independent manner and bear the CD5 marker. The proliferation of this B cell population may be the host's response to the attempt by the virus to circumvent the immune response by complexing with host lipoproteins. It is proposed that HCV complexed to VLDL is the antigen that directly stimulates the proliferation of these primordial type B cells. Testing of these hypotheses may produce insights not only into the etiology of mixed cryoglobulinemia but possibly into the mechanisms by which HCV circumvents the immune response and established chronic infection.

Mixed cryoglobulinemia secondary to hepatitis C virus infection.

Mixed cryoglobulinemia is a systemic vasculitis with clinical manifestations ranging from the characteristic benign-appearing syndrome of palpable purpura, arthrologies, and fatigue to severe vasculitis involving vital organs. A strong association of the disease with hepatitis C virus infection and the demonstration of the specific concentration of the virus in the cryoglobulins have implicated hepatitis C virus in the etiopathogenesis of the disease. The increase in illicit intravenous drug use in the past 30 years seems to have raised the occurrence in the United States of this once uncommon disease and changed the demographics: there seem to be more male intravenous drug users in their forties with the disease than women without risk factors for hepatitis C virus infection in their fifties and sixties. Pathogenesis, therapy, and the hypothesis on the etiologic role of hepatitis C virus are reviewed, and the implications of recent studies and new concepts for treatment of this often benign-appearing but deceptive and potentially life-threatening disease are discussed.

The association of hepatitis C virus infection with monoclonal rheumatoid factors bearing the WA cross-idiotype: implications for the etiopathogenesis and therapy of mixed cryoglobulinemia.

OBJECTIVE: To determine the prevalence of monoclonal rheumatoid factors (mRF) bearing the WA cross-idiotype (WA XId) in hepatitis C virus (HCV) positive type II mixed cryoglobulins, to review recent studies on the role of HCV in the cutaneous vasculitis lesions in patients with type II cryoglobulinemia and to discuss the implication of these studies for the etiopathogenesis and therapy of the disease. METHODS: Thirty type II cryoglobulins were tested for WA and PO XId and for HCV RNA: RESULTS: WA mRF were strongly, although not exclusively, associated with HCV in type II mixed cryoglobulinemia. CONCLUSION: These and other recent studies from our laboratory suggest that chronic HCV infection may be the stimulus for the production of WA mRF and that HCV may be directly involved in the pathogenesis of the cutaneous vasculitis in patients with type II cryoglobulinemia. The association of HCV infection with the disease provides a rationale for anti-viral therapy and for monitoring therapy by measuring the HCV level in both blood and liver.

The aetiology of mixed cryoglobulinaemia associated with hepatitis C virus infection.

A strong association of hepatitis C infection (HCV) with 'essential' mixed cryoglobulinaemia has been established. The demonstration of HCV in Type II mixed cryoglobulins with monoclonal rheumatoid factors (mRF) that bear the WA crossidiotype has lead to the hypothesis that mixed cryoglobulins result from chronic stimulation by HCV-lipoprotein of a population of XId WA+B-1a cells. The reactivity of WA IgM initially produced is with the HCV-self antigen complex with RF activity resulting secondarily from the pausi-mutational process accompanying the T cell independent process. This benign proliferation progresses by multi step mutations to malignancy in a minority of patients. The implications of the hypothesis for understanding the physiology of certain natural auto antibodies and for therapeutic intervention in this disease are discussed.