Many microbes, toxins, autoimmune diseases, and neoplastic diseases may cause liver inflammation; however, 5 viruses whose main pathogenesis is liver disease are referred to as hepatitis A, B, C, D, and E viruses. These viruses cause a significant burden of global illness. With the exception of hepatitis A virus, all may cause chronic infection potentially leading to cirrhosis and hepatocellular carcinoma. Excellent serologic and nucleic acid detection methods are available for determining the precise cause and, in some cases, the duration of infection. Diagnostics are critical for identifying individuals needing treatment and for monitoring the treatment success.

The criterion standard for the diagnosis of occult hepatitis C virus (HCV) infection is detection of HCV-RNA in liver cells. However, because of the invasive nature of liver biopsy, other methods have been studied. The present study aimed to identify subjects with occult HCV-4 infection among healthy sexual partners of patients with chronic HCV-4 infection by detecting HCV-RNA in peripheral blood mononuclear cells (PBMCs) using real-time polymerase chain reaction (PCR). Fifty healthy Egyptian spouses of patients with chronic HCV-4 infection were included in this study. Real-time PCR was used to detect HCV-RNA in PBMCs in all the study subjects. The prevalence of occult HCV-4 infection was 4%, and a statistically significant higher prevalence was found among patients with a history of sexually transmitted infection. The results of the present study indicate the importance of intra-spousal transmission of HCV-4 infection, especially in subjects with a history of sexually transmitted infection.

Mixed cryoglobulinemia is the most common extrahepatic disease manifestation of chronic hepatitis C virus (HCV) infection, where immunoglobulins precipitate at low temperatures and cause symptoms such as vasculitis, glomerulonephritis and arthralgia. HCV-associated cryoglobulinemia is also strongly linked with the development of B cell non-Hodgkin lymphoma. Abnormal B cell function in HCV infections can lead to the formation of HCV cryoglobulin complexes that usually comprise monoclonal rheumatoid factor and HCV-specific immune complexes. The aim of this study was to characterize the activation phenotype of B cells from patients with chronic HCV infection in comparison to healthy controls using flow cytometry. In addition, we determined how the activation status varies depending on the presence of cryoglobulinemia and advanced liver fibrosis. We found that only memory B cells, not naïve cells, were significantly activated in chronic HCV infection when compared with healthy controls. We also identified markers of memory B cell activation that were specific for HCV patients with cryoglobulinemia (CD86, CD71, HLA-DR) and advanced liver disease (CD86). Our results demonstrate that HCV infection has differential effects on B cells depending on the severity of hepatic and extrahepatic disease.

Mixed cryoglobulinemia (MC) is a syndrome resulting from cold-insoluble immunoglobulin complexes or cryoglobulins (CGs) that precipitate in the serum of 40% to 50% of patients with chronic hepatitis C virus (HCV) infection. The pathogenesis of cryoglobulinemia likely occurs due to chronic viremia and generation of rheumatoid factor following continuous presentation of antigen-immunoglobulin complexes to B cells. CGs are thought to be responsible for a variety of extrahepatic manifestations associated with HCV, including vasculitis, glomerulonephritis, arthritis, and neuropathies, which occur in approximately 10% of HCV patients with CGs. CGs also are a powerful predictive factor for progressive liver disease and the aggressive reoccurrence of liver disease in HCV-positive patients after liver transplantation. First-line therapy for MC due to HCV infection is antiviral therapy with pegylated interferon-alpha and ribavirin. Viral eradication usually produces marked reduction of physical complications and arrests end organ damage concomitant with clearance of CG. Additional prospective, controlled studies are necessary to determine whether CG influences patient virologic response and/or its durability to antiviral therapy. Immunomodulators such as corticosteroids and cyclophosphamide are efficacious for palliative treatment of the symptomatology of HCV cryoglobulinemia but may enhance viral replication. Consequently, prolonged therapy with immunomodulatory agents should be limited to severe vasculitis or aggressive glomerulonephritis in patients with MC due to HCV who have failed to respond to antiviral therapy. In acute, fulminant presentations, plasmapheresis may provide temporary relief and arrest the rapid progression of the disease so that additional therapy can be initiated.

Patients with chronic hepatitis C virus (HCV) infection frequently have many extrahepatic manifestations, as persistent HCV infection often triggers lymphoproliferative disorders and metabolic abnormalities. These manifestations primarily include autoimmune disorders such as cryoglobulinemia, Sjögren's syndrome, and autoimmune thyroid disorders. It has been well established that chronic HCV infection plays important roles in the production of non-organ-specific autoantibodies, including antinuclear antibodies and smooth muscle antibodies, and organ-specific autoantibodies such as thyroid autoantibodies. However, the clinical significance of autoantibodies associated with the extrahepatic manifestations caused by HCV infection has not been fully recognized. In this paper, we mainly focus on the relationship between extrahepatic manifestations and the emergence of autoantibodies in patients with HCV infection and discuss the clinical relevance of the autoantibodies in the extrahepatic disorders.

Patients with chronic hepatitis C virus (HCV) infection frequently have many extrahepatic manifestations, as persistent HCV infection often triggers lymphoproliferative disorders and metabolic abnormalities. These manifestations primarily include autoimmune disorders such as cryoglobulinemia, Sjögren's syndrome, and autoimmune thyroid disorders. It has been well established that chronic HCV infection plays important roles in the production of non-organ-specific autoantibodies, including antinuclear antibodies and smooth muscle antibodies, and organ-specific autoantibodies such as thyroid autoantibodies. However, the clinical significance of autoantibodies associated with the extrahepatic manifestations caused by HCV infection has not been fully recognized. In this paper, we mainly focus on the relationship between extrahepatic manifestations and the emergence of autoantibodies in patients with HCV infection and discuss the clinical relevance of the autoantibodies in the extrahepatic disorders.

Patients with chronic hepatitis C virus (HCV) infection frequently have many extrahepatic manifestations, as persistent HCV infection often triggers lymphoproliferative disorders and metabolic abnormalities. These manifestations primarily include autoimmune disorders such as cryoglobulinemia, Sjögren's syndrome, and autoimmune thyroid disorders. It has been well established that chronic HCV infection plays important roles in the production of non-organ-specific autoantibodies, including antinuclear antibodies and smooth muscle antibodies, and organ-specific autoantibodies such as thyroid autoantibodies. However, the clinical significance of autoantibodies associated with the extrahepatic manifestations caused by HCV infection has not been fully recognized. In this paper, we mainly focus on the relationship between extrahepatic manifestations and the emergence of autoantibodies in patients with HCV infection and discuss the clinical relevance of the autoantibodies in the extrahepatic disorders.

Impaired autonomic function has been described in patients with chronic liver diseases from different aetiologies, and has proven to be a poor prognostic indicator. To date, it is not known how chronic hepatitis C virus (HCV) infection affects the autonomic nervous system.

In the present study, we compared cardiovagal autonomic function in patients with chronic HCV infection and healthy controls and examined the relation between autonomic function and serum levels of aminotransferases, HCV RNA, cryoglobulins, albumin and glucose.

Autonomic function was assessed in 45 treatment-naïve patients with chronic HCV infection and in 40 healthy controls by determining spontaneous baroreflex sensitivity (BRS) and heart rate variability (HRV) indices. The R-R interval was determined by electrocardiogram recording; continuous radial artery pressure was monitored simultaneously by applanation tonometry. Laboratory analyses and quantitative polymerase chain reaction for serum HCV RNA level were performed by standard procedures.

BRS and HRV time and frequency domain indices were lower in patients with HCV infection compared with healthy controls [7.1+/-3.4 vs. 11.5+/-6.5 ms/mmHg for BRS, 168.5+/-160.9 vs. 370.7+/-349.4 ms(2) for low-frequency HRV (mean+/-SD); P<0.01]. Multivariate analysis showed that autonomic dysfunction in HCV-infected patients correlated with elevated alanine aminotransferase levels, but was not associated with serum HCV RNA levels and cryoglobulins.

Our results suggest that impaired autonomic function is caused by chronic HCV infection. Further studies are needed, however, to identify the underlying mechanisms.

Mixed cryoglobulinemia (MC) has a strong association with hepatitis C virus (HCV) infection and is associated with a higher degree of fibrosis and poor response to therapy. Currently, there are no known histological findings on liver biopsy that correlate with the presence of MC in HCV-infected patients, although we have occasionally noted prominent sinusoidal lymphocytosis in MC patients. The goal of this study is to determine whether sinusoidal lymphocytosis is a histological marker of MC in liver biopsies from patients with chronic hepatitis C. The liver clinic database at our institution was searched for chronic hepatitis C patients with MC who underwent liver biopsy during 1999-2005. Ten such cases were identified and were included in the study. Ten chronic hepatitis C MC-negative cases were matched for age and stage of fibrosis to serve as controls. Histological features (sinusoidal lymphocytes, inflammatory activity, acidophil bodies, and fibrosis stage) were evaluated in each biopsy. Clinical and laboratory data (serum protein electrophoresis, liver enzymes, hepatitis C viral load, treatment status, comorbidities, etc.) were also recorded. Formalin-fixed, paraffin-embedded sections were submitted for immunohistochemical analysis using antibodies against CD3, CD20, CD4, CD8, and CD68. Sinusoidal lymphocytes were counted in 5 hpf (40x) on hematoxylin and eosin (H&E) stain, and on CD3 and CD20 immunostains. The number of CD68+ Kupffer cells was also counted in a similar fashion. In the MC-positive versus MC-negative cases, mean fibrosis stage (2.4 vs. 2.4), inflammatory grade (1.7 vs. 2.1), lymphocyte count (359 vs. 128/5 hpf), and Kupffer cell count (239 vs. 220/5 HPF) were assessed. There was a significant increase in sinusoidal T-cell lymphocytes (P < 0.05) in MC-positive cases as compared to MC-negative cases. Nearly all sinusoidal lymphocytes were CD8-positive cells in both groups. Other histological parameters did not differ in the two groups. MC-positive cases tended to have a lower viral load as compared to controls (P= 0.059). The role of sinusoidal T cells in the pathogenesis of MC is currently unknown. It is unclear if the presence of these cells implies ongoing antigenic stimulation that may lead to increased risk of lymphoma. This feature may be an important clue to predict the presence of MC, an HCV-associated phenomenon that has important implications for response to treatment and disease progression.

Hepatitis C virus (HCV) is a hepatotropic virus causing hepatocellular damage and chronic liver inflammation that progressively can lead to cirrhosis and hepatocellular carcinoma (HCC). HCV is also lymphotropic, as demonstrated by its capacity to replicate in lymphocytes, by the recurrent detection of organ- and non-organ-specific autoantibodies in HCV-infected patients, and by the strong association found between HCV infection and type II mixed cryoglobulinemic syndrome (MC-II). Moreover, accumulating data ascribe an etiopathogenetic role in the development of B cell non-Hodgkin's lymphomas (NHL) to HCV. All these findings account for the profound effect of HCV infection in the host's immune system. The unique virus-host interactions that culminate in the generation and sustained production of autoantibodies and cryoglobulins have not been delineated. It appears that chronic antigenic stimulation could cause the emergence of specific B cell clones that produce cryoglobulins; moreover, B cell activation and/or deregulation could originate as a result of HCV binding to CD81 tetraspanin or as a consequence of its ability to replicate in B cells. In a previous study we demonstrated that, in MC-II HCV-positive patients, cryoprecipitated monoclonal IgMs, and B cell receptors (BCR) of overexpanded B cell clones share the same combinatory region. Moreover, these IgMs were reactive against both the Fc region of human IgG and the HCV-NS3 antigen. NS3 and Fc epitopes have been idengified by epitope excision approach. One of the idengified NS3 epitopes has been used to immunize a mouse and the monoclonal antibody obtained showed the same cross-reactivity as patients' IgMs. The characterization of antigenic specificity of this antibody may be useful to idengify antigens that can stimulate B cell proliferation in HCV-infected individuals.

In hepatitis C virus (HCV)-positive liver transplant recipients, infection of the allograft and recurrent liver disease are important problems. Increased donor age has emerged as an important variable affecting patient and graft survival; however, specific age cutoffs and risk ratios for poor histologic outcomes and graft survival are not clear.

A longitudinal database of all HCV-positive patients transplanted at our center during an 11-year period was used to identify 111 patients who received 124 liver transplants. Graft survival and histological endpoints (severe activity and fibrosis) of HCV infection in the allografts were compared as a function of donor age at transplantation.

By Kaplan-Meier analyses, older allografts showed earlier failure and decreased time to severe histological activity and fibrosis as compared with allografts from younger donors. By Cox proportional hazards analysis, older allografts were at greater risk for all severe histologic features and decreased graft survival as compared with younger allografts (P< or =0.02 for all outcomes). Analysis of donor age as a dichotomous variable showed that donors greater than 60 yr were at high risk for deleterious histologic outcomes and graft failure. An age cutoff of 60 yr showed a sensitivity of 94% and specificity of 67% for worse graft survival by receiver operating characteristics curve.

Advanced donor age is associated with more aggressive recurrent HCV and early allograft failure in HCV-positive liver transplant recipients. Consideration of donor age is important for decisions regarding patient selection, antiviral therapy, and organ allocation.

Occult hepatitis C virus (HCV) infection is a new recently characterized entity. This occult infection can be present in two different clinical situations: in anti-HCV negative, serum HCV-RNA negative patients with abnormal liver function tests and in anti-HCV positive subjects with normal values of liver enzymes and without serum HCV-RNA. This review describes recent studies of occult HCV infection in both kinds of patients.

Chronic hepatitis C virus (HCV) infection causes cirrhosis in many infected patients; however, a better understanding of the risk factors for fibrosis progression in high HCV prevalence groups such as US veterans is needed. We wished to compare the demographic, clinical characteristics, and independent variables that influence fibrosis in US veterans vs nonveterans with chronic HCV. HCV-seropositive US veterans (n = 459) and nonveterans (n = 395) prospectively completed a detailed medical, social and occupational questionnaire. Clinical factors for progressive liver disease were compared between veterans and nonveterans and fibrosis stage assessed on liver biopsies (168 veterans and 208 nonveterans). Using polychotomous logistic regression, fibrosis was analysed as both a progressive and categorical outcome to determine independent risk factors for both patient groups. Although veterans were significantly older and had higher lifetime alcohol consumption than nonveterans, their median fibrosis scores did not differ from nonveterans. By univariate analysis, alanine aminotransferase, necroinflammatory activity (NIA), and cryoglobulin positivity were associated with fibrosis in veterans and nonveterans (P < 0.05, all comparisons), whereas steatosis was associated with fibrosis only in nonveterans (P < 0.0001). By multivariate analysis, NIA was an independent risk factor for fibrosis in both groups (P < 0.01). However, fibrosis in nonveterans was also independently associated with steatosis, significant alcohol consumption and age (P < 0.04, all comparisons). Independent risk factors for fibrosis vary among high HCV prevalence groups such as veterans when compared with nonveterans. Understanding specific patient cohort effects is important for determining independent risk factors for disease progression in chronic HCV infection.

In this study we aimed to determine whether serum B-lymphocyte activating factor (BAFF) level is increased in patients with chronic hepatitis C virus (HCV) infection, and to assess its association with HCV-related autoimmunity. Sixty-five patients with chronic HCV infection were compared with two disease control groups [57 patients with systemic lupus erythematosus (SLE) and 15 with chronic hepatitis B virus (HBV) infection] and a healthy control group of 35 individuals. A special attention was given to HCV-related arthralgia and or vasculitis. Serum BAFF was assessed in all studied individuals, whereas rheumatoid factor (RF), anti-cardiolipin antibodies (aCL), and cryoglobulins were determined in HCV and HBV infected patients, and anti-dsDNA antibodies and aCL were assessed in patients with SLE. Mean serum BAFF was increased in patients with HCV infection and SLE (2.4+/-0.8 ng/ml and 3.1+/-1.34 ng/ml respectively) compared to 1.1+/-0.14 ng/ml in patients with HBV; and to 1.1+/-0.27 in healthy controls (all, p<0.0001). The elevation in serum BAFF was associated with HCV-related arthralgia and or vasculitis (p<0.0001), and with the presence of aCL and of cryoglobulins. HBV patients lacked features suggestive of autoimmunity. In SLE patients, elevated serum BAFF was in association with the presence of anti-dsDNA (p=0.002). As in other autoimmune diseases, increased serum BAFF was also found in patients with chronic HCV infection. Elevated serum BAFF levels were associated with clinical and laboratory features of autoimmunity, suggesting that BAFF may play a role in HCV-related autoimmunity.

Chronic hepatitis C virus (HCV) infection is frequently associated with a number of extrahepatic complications. In the majority of cases the underlying pathogenetic mechanisms are immune mediated, as evidenced by the presence of circulating autoantibodies (mixed cryoglobulinemia), whereas for others a localized host cellular immune response is implicated (e.g. sialadenitis, lichen planus). In this review, the latest data on the pathogenesis and clinical manifestations of the most common autoimmune extrahepatic manifestations of chronic HCV infection are presented.

We designed two synthetic-core-specific peptides core 1 (C1) and core 2 (C2), and an E1-specific peptide (E1). We produced specific polyclonal antibodies against these peptides and used the antibodies for detection of HCV antigens on surface and within infected peripheral blood leukocytes.

Peripheral blood from a healthy individual who tested negative for HCV RNA was incubated with HCV type 4 infected serum for 1 h and 24 h at 37 degrees C. Cells were stained by direct and indirect immunofluorescence and measured by flow cytometry.

After 1 h of incubation, antibodies against C1, C2, and E1 detected HCV antigens on the surface of 27%, 26% and 73% of monocytes respectively, while 10%, 5% and 9% of lymphocytes were positive with anti-C1, anti-C2 and anti-E1 respectively. Only 1-3% of granulocytes showed positive staining with anti-C1, anti-C2 and anti E1 antibodies. After 24 h of incubation, we found no surface staining with anti-C1, anti-C2 or anti-E1. Direct immunostaining using anti-C2 could not detect intracellular HCV antigens, after 1 h of incubation with the virus, while after 24 h of incubation, 28% of infected cells showed positive staining. Only plus strand RNA was detectable intracellularly as early as 1 h after incubation, and remained detectable throughout 48 h post-infection. Interestingly, minus RNA strand could not be detected after 1 h, but became strongly detectable intracellularly after 24 h post-infection.

Monocytes and lymphocytes are the preferred target cells for HCV infection in peripheral blood leukocytes. Our specific anti-core and anti-E1 antibodies are valuable reagents for demonstration of HCV cell cycle. Also, HCV is capable of infecting and replicating in peripheral blood mononuclear cells as confirmed by detection of minus strand HCV RNA as well as intracellular staining of core HCV antigen.

We designed two synthetic-core-specific peptides core 1 (C1) and core 2 (C2), and an E1-specific peptide (E1). We produced specific polyclonal antibodies against these peptides and used the antibodies for detection of HCV antigens on surface and within infected peripheral blood leukocytes.

Peripheral blood from a healthy individual who tested negative for HCV RNA was incubated with HCV type 4 infected serum for 1 h and 24 h at 37 degrees C. Cells were stained by direct and indirect immunofluorescence and measured by flow cytometry.

After 1 h of incubation, antibodies against C1, C2, and E1 detected HCV antigens on the surface of 27%, 26% and 73% of monocytes respectively, while 10%, 5% and 9% of lymphocytes were positive with anti-C1, anti-C2 and anti-E1 respectively. Only 1-3% of granulocytes showed positive staining with anti-C1, anti-C2 and anti E1 antibodies. After 24 h of incubation, we found no surface staining with anti-C1, anti-C2 or anti-E1. Direct immunostaining using anti-C2 could not detect intracellular HCV antigens, after 1 h of incubation with the virus, while after 24 h of incubation, 28% of infected cells showed positive staining. Only plus strand RNA was detectable intracellularly as early as 1 h after incubation, and remained detectable throughout 48 h post-infection. Interestingly, minus RNA strand could not be detected after 1 h, but became strongly detectable intracellularly after 24 h post-infection.

Monocytes and lymphocytes are the preferred target cells for HCV infection in peripheral blood leukocytes. Our specific anti-core and anti-E1 antibodies are valuable reagents for demonstration of HCV cell cycle. Also, HCV is capable of infecting and replicating in peripheral blood mononuclear cells as confirmed by detection of minus strand HCV RNA as well as intracellular staining of core HCV antigen.

Recurrent hepatitis C virus (HCV) infection in patients after liver transplantation is an important clinical problem. Because serum cryoglobulins (CG) are known to be associated with an increased incidence of cirrhosis in nontransplant patients, the authors tested the hypothesis that CG would also predict aggressive recurrent HCV in patients after liver transplantation.

Using a longitudinal database, the outcomes of 105 allografts transplanted into 97 HCV-positive patients from 1991 through 2002 were analyzed on the basis of CG status using a retrospective cohort design. Fifty-nine CG-negative and 38 CG-positive patients were identified. Histologic outcomes and graft survival were analyzed using Kaplan-Meier estimates and Cox univariate and multivariate analyses. Both overall survival and HCV-specific survival (non-HVC-related deaths and graft losses censored) were analyzed.

By Kaplan-Meier estimates, CG-positive patients showed earlier graft failure with decreased time to severe histologic activity and fibrosis as compared with CG-negative patients (P<0.05 for all outcomes). By univariate analysis, CG-positive patients had significantly higher risk ratios for shortened HCV-specific graft survival, severe activity-free survival, and severe fibrosis-free survival as compared with CG-negative patients (P<0.05 for all outcomes). In the multivariate model, CG was an independent predictor for severe activity-free, severe fibrosis-free, and HCV-specific graft survival (P<0.05 for all outcomes).

CG-positivity is associated with severe recurrent HCV disease in liver transplant recipients.

The existence of an extrahepatic hepatitis C virus replication compartment is an important question for optimizing therapy and preventing the infection of liver grafts. An extraheptic replication compartment could be indicated if viral decline during the anhepatic phase is not a single exponential. However, the duration of the anhepatic phase is too short (0.5-2h) to allow such analysis. Here we mathematically analyze viral decline during liver transplantation beyond the period of the anhepatic phase and examine the possibility of viral compartmentalization.

Viral load of 30 patients undergoing liver transplantation was frequently measured. Simulation and non-linear fitting of differential equation models were used to test different compartmentalization hypotheses.

In 16 of the patients (56%), a bi-phasic viral decline was observed which is explained by the existence of a second replication compartment. This extrahepatic compartment is responsible for about 3.1% of virus in circulation and the mean half-life of its infected cells is 2.6 days. The remaining patients, with a single exponential decline, have either a second compartment with relatively low contribution or no second compartment.

These results provide a first quantitative picture of the extrahepatic hepatitis C viral contribution and may suggest new approaches for viral clearance.

Routine analysis of serum and/or plasma specimens for hepatitis C virus (HCV) RNA does not always correctly reflect the response to antiviral therapy. Analysis of whole-blood specimens for detection of viral RNA should provide more-accurate prognostic information.

Whole-blood, serum, and plasma specimens (268 sample sets) were obtained from 56 patients who did not respond to initial interferon (IFN)- alpha 2b monotherapy (5 MU every 2 days for 3 months). Specimens were analyzed for HCV RNA by 4 different types of reverse-transcriptase polymerase chain reaction (RT-PCR) (Cobas Amplicor HCV-2.0 [Roche], LightCycler real-time PCR [Roche], and 2 in-house RT-PCRs) to determine whether specimen type can predict the rate of virologic response to high-dose treatment with IFN (10 MU every 2 days) and ribavirin (1-1.2 g/day).

Of the 56 patients who provided specimens, serum and plasma obtained from 18 tested negative for HCV RNA at the end of treatment, indicating a complete virologic response. In contrast, analysis of whole-blood specimens obtained at the same time revealed the presence of viral RNA in 12 of these 18 patients. All 12 subjects had relapse of HCV in serum and plasma: 11 relapsed a median of 4 weeks after the end of treatment, and 1 relapsed 20 weeks after the end of treatment. None of these 12 patients--all of whom consistently had whole-blood specimens that tested positive and plasma and serum specimens that tested negative for HCV RNA up to 20 weeks before the end of treatment--showed a sustained virologic response (P=.0002).

Results of whole-blood tests for detection of HCV RNA were highly predictive of viral relapse (positive predictive value, 100%) and thus may be useful tools for monitoring and tailoring IFN/ribavirin therapy. Testing of only serum or plasma specimens underestimates the true circulating HCV load and leads to an overestimation of antiviral response rates.

Mixed cryoglobulinemia (MC) is the most common extrahepatic manifestation of HCV infection. The aim of this study is to determine the prevalence of MC in HCV infected Greek patients and to identify if it is associated with liver histology or the mode of HCV transmission.

One hundred and twenty-six patients with chronic HCV infection were evaluated for the presence of serum cryoglobulins, autoantibodies and viral markers. One hundred and eighteen of them underwent liver biopsy and each specimen was evaluated according to the grading and staging system described by Ishak et al.

Cryoglobulins were detected in 37/126 (29.4%) HCV patients and cryocrit values ranged between 0.5 and 6.5%. Only two patients presented clear clinical manifestations of MC. In patients with MC, a higher grading (6.40+/-2.06 vs. 5.27+/-2.55, p=0.013) and staging score (3.71+/-1.45 vs. 2.83+/-1.84, p=0.007) was noted in liver biopsy compared to those without MC. Logistic regression analysis identified staging score (OR, 1.33; CI, 1.06-1.66, p=0.015) as the only independent variable associated with cryoglobulinemia. Correlation between the presence of cryoglobulins and the mode of HCV transmission was not found.

Greek patients with chronic HCV infection have high prevalence of cryoglobulinemia. A clear association between the presence of serum cryoglobulins and staging score of chronic hepatitis was found, with no difference in patients' age or the duration of infection. It is possible that cryoglobulinemia results in more rapid hepatic fibrosis in HCV infected patients.

Mixed cryoglobulinaemia, when not secondary to other well-defined immunological disorders, is commonly associated with hepatitis C virus (HCV) infection. However, a minority of cases lack evidence of HCV infection and are, therefore, defined as 'true essential' mixed cryoglobulinaemias. We thoroughly investigated three such patients to determine the aetiology of this disorder. Antibodies to HCV (anti-HCV) and HCV RNA, detected by sensitive enzyme-linked immunosorbent and polymerase chain reaction assays in serum and in concentrated cryoglobulins, were repeatedly negative in the three patients. Despite the lack of evidence for HCV infection, two of them were still treated with interferon alpha-2a assuming unrecognized viral infection. Both patients demonstrated excellent clinical and laboratory responses, but cryoglobulinaemia relapsed after the withdrawal of therapy. At the time of relapse, HCV RNA genomic sequences were detected for the first time in the cryoprecipitates of both patients. In the third case, HCV RNA was demonstrated for the first time during a flare of cryoglobulinaemia coincident with varicella infection. In all three patients anti-HCV antibodies remained negative throughout follow-up. We conclude that some apparently 'essential' forms of mixed cryoglobulinaemia can be caused by occult HCV infection. Interferon therapy can be taken into consideration in such HCV-negative cases.

Although hepatitis C virus (HCV) is a recognized cause of circulating cryoglobulins, the role of human immunodeficiency virus (HIV) in the pathogenesis of cryoglobulinemia has not been investigated extensively. To evaluate the prevalence of circulating cryoglobulins and to assess the relationship with clinical and virological parameters, 162 HIV-positive subjects (84 anti-HCV(+)) were tested for cryoglobulins, C3, C4, RF, autoantibodies, HIV-viral titer, and CD4(+) count. Anti-HCV-positive subjects were tested for HCV-RNA, HCV-viral titer, and HCV genotype. All patients were examined for the presence of signs or symptoms of vasculitis and tested for cryoglobulins using a standard biochemical assay. Cryoglobulins were found in 30 (18.5%) cases. Of the 30 positive cases, 29 (96.7%) were anti-HCV-positive and 28 (93.3%) HCV-RNA-positive. The presence of cryoglobulins was significantly associated (P < 0.01) with HCV-RNA positivity (OR = 27), liver cirrhosis (OR = 16), decreased levels of C3 (OR = 8.6), C4 (OR = 13.6), increased levels of IgG and IgM (OR = 6.1 and 7.9, respectively), and RF positivity (OR = 6.3), but was unrelated to CD4(+) cell count, HIV viral load, diagnosis of AIDS, HCV viral load and the presence of autoantibodies. Interestingly, the presence of cryoglobulins was not significantly associated with signs and symptoms commonly associated with cryoglobulinemia. In conclusion, HIV infection does not seem to play a significant role in the production of circulating cryoglobulins, which strongly correlates with HCV co-infection and liver cirrhosis. Typical signs and symptoms of cryoglobulinemia do not correlate with the detection of circulating cryoglobulins in HIV and HCV patients.

We used a baculovirus-based system to prepare structural proteins of hepatitis C virus (HCV) genotype 1a. Binding of this preparation to cultured human hepatic cells was both dose dependent and saturable. This binding was decreased by calcium depletion and was partially prevented by ligands of the asialoglycoprotein receptor (ASGP-R), thyroglobulin, asialothyroglobulin, and antibody against a peptide in the carbohydrate recognition domain of ASGP-R but not preimmune antibody. Uptake by hepatocytes was observed with both radiolabeled and dye-labeled HCV structural proteins. With hepatocytes expressing the hH1 subunit of the ASGP-R fused to green fluorescent protein, we could show by confocal microscopy that dye stain cointernalized with the fusion protein in an area surrounding the nucleus. Internalization was more efficient with a preparation containing p7 than with one that did not. The two preparations bound to transfected 3T3-L1 cells expressing either both (hH1 and hH2) subunits of the ASGP-R (3T3-22Z cells) or both hH1 and a functionally defective variant of hH2 (3T3-24X cells) but not to parental cells. Additionally, uptake of dye-labeled preparation containing p7 was observed with 3T3-22Z cells but not with 3T3-L1 or 3T3-24X cells or with the preparation lacking p7, suggesting that p7 regulates the internalization properties of HCV structural proteins. Our observations suggest that HCV structural proteins bind to and cointernalize with the ASGP-R in cultured human hepatocytes.

Approximately 40% of patients with chronic hepatitis C virus (HCV) infection develop detectable serum cryoglobulins or cryoprecipitates (CP), although most do not show clinical or physical signs of syndromic cryoglobulinemia. Although association of HCV with the extrahepatic complications of cryoglobulinemia is widely recognized, the relationship of cryoglobulinemia with liver disease is unclear. We wished to study the relationship between CP and cirrhosis and to determine whether the development of CP is a true covariate for progressive liver disease or a confounding variable that impacts cirrhosis because of patient age, duration of disease, or differences in gender. We undertook a meta-analysis of 19 studies published between 1994 and 2001. The incidence of cirrhosis was compared in patients with and without CP after logistic regression adjustments for accepted risk factors for progressive liver disease, including age, gender, and estimated duration of disease (EDD). A total of 2,323 patients with chronic hepatitis C were identified, with 1,022 (44%) having detectable CP. Cirrhosis was present in 40% of patients with CP but only 17% of patients without CP (total Chi;(2) = 141.69, P <.001). After adjusting for age, gender, and estimated duration of disease by logistic regression, the combined odds ratio for incidence of cirrhosis in patients CP positive versus CP negative was 4.87, (95% CI: 3.32, 7.15), indicating a highly significant association between cirrhosis and cryoglobulinemia. In conclusion, cryoglobulins may be a useful prognostic indicator for increased risk of cirrhosis with chronic hepatitis C.

Hepatitis C virus-like particles (HCV-LPs) containing the structural proteins of HCV H77 strain (1a genotype) was used as a model for HCV virion to study virus-cell interaction. HCV-LPs showed a buoyant density of 1.17 to 1.22 g/cm(3) in a sucrose gradient and formed double-shelled particles 35 to 49 nm in diameter. Flow cytometry analysis by an indirect method (detection with anti-E2 antibody) and a direct method (use of dye-labeled HCV-LPs) showed that HCV-LPs binds to several human hepatic (primary hepatocytes, HepG2, HuH7, and NKNT-3) and T-cell (Molt-4) lines. HCV-LPs binding to cells occurred in a dose- and calcium-dependent manner and was not mediated by CD81. Scatchard plot analysis suggests the presence of two binding sites for HCV-LPs with high (K(d) approximately 1 microg/ml) and low (K(d) approximately 50 to 60 microg/ml) affinities of binding. Anti-E1 and -E2 antibodies inhibited HCV-LPs binding to cells. While preincubation of HCV-LPs with very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), or high-density lipoprotein (HDL) blocked its binding to cells, preincubation of cells with VLDL, LDL, HDL, or anti-LDL-R antibody did not. Confocal microscopy analysis showed that, after binding to cells, dye-labeled HCV-LPs were internalized into the cytoplasm. This process could be inhibited with anti-E1 or anti-E2 antibodies, suggesting that E1 and E2 proteins mediate HCV-LPs binding and, subsequently, their entry into cells. Altogether, our results indicate that HCV-LPs can be used to further characterize the mechanisms involved in the early steps of HCV infection.

Hepatitis C virus (HCV) infection is associated with immune-mediated abnormalities and B-cell lymphoproliferation evolving to an overt lymphoma. Recently, CD81 was identified as an HCV receptor on B-lymphocytes, providing a mechanism by which B cells are infected and activated by the virus. In addition, expansion of CD5+ B lymphocytes was described to be associated with various non-HCV related autoimmune disorders. Therefore, we studied the possible role of peripheral B cells CD81 and CD5 over-expression in the development of HCV-related autoimmunity and their association with disease severity in chronic HCV infection. Peripheral B cells CD5 expression and mean fluorescence intensity (MFI) of CD81 were determined in 30 HCV-infected patients, 30 healthy controls and 15 patients with hepatitis B virus infection using fluorescence-activated cell scan (FACS). We have also investigated the association between peripheral CD5 and CD81 B-cell over-expression and markers of autoimmunity and disease severity in patients chronically infected by HCV. CD5+ B-cells were increased in chronic HCV infection (23.2 +/- 7.2%) compared with those of healthy controls (15 +/- 5.5%) (P < 0.0001) and chronic HBV infection (19 +/- 3.7%) (P = 0.08). CD81 MFI was significantly higher in HCV-infected compared to HBV-infected patients and healthy controls. Both increased CD81 MFI and CD5+ B-cell expansion were associated with the production of rheumatoid factor and mixed cryoglobulins and positively correlated with HCV viral load and histological activity index. The overexpression of CD81 and the expansion of CD5+ peripheral B-cells in HCV-infected patients may possibly play a role in the development of HCV-associated autoimmunity and lymphoproliferation.

When TT virus (TTV) DNA was quantitated in whole blood and plasma aliquots from 27 viremic individuals by real-time detection PCR that can detect essentially all TTV genotypes, the TTV load was 6.9 +/- 3.5 (mean +/- standard deviation)-fold higher in the whole blood than in the plasma samples [P < 0.002 (paired t test)]. To clarify the reason for this difference, peripheral blood cells of various types including red blood cells, granulocytes (CD15+), B cells (CD19+), T cells (CD3+), monocytes (CD14+), and NK cells (CD3-/CD56+) were separated at a purity of 95.4-99.5% from each of three infected individuals with relatively high TTV viremia, and their TTV viral loads were determined. Red blood cells were uniformly negative, but the other cell types were positive for TTV DNA at various titers. In all three patients, the highest TTV load was found in granulocytes (4.2 x 10(4)-3.1 x 10(5) copies/10(6) cells), followed by monocytes (1.4-2.2 x 10(4) copies/10(6) cells) and NK cells (5.4-6.5 x 10(3) copies/10(6) cells); B and T cells were positive, with a low viral load (6.7 x 10(1)-2.7 x 10(3) copies/10(6) cells). These results indicate that TTV is distributed in various peripheral blood cell types at distinct levels, with the highest viral load in granulocytes, and that a significant proportion of the TTV DNA in peripheral blood is not identified by the standard plasma/serum DNA detection methods.

The overall role of complement in the host--pathogen relationship is now well understood. However, its involvement at a chronic stage of infection, such as chronic hepatitis C, is less well documented. Here, results are reported which point to the use of specific C4 monitoring in the follow-up of HCV patients. This study concerns 66 patients with chronic HCV infection, treated with interferon alpha 2b alone or with interferon alpha 2b + ribavirin, and 50 healthy adults as controls. Complement blood tests were performed to measure C1q, C3, C4, mannan binding lectin (MBL), C1s-C1 inhibitor complexes, total (CH50) and C4 (C4H) haemolytic activity; specific C4 activity was taken as the C4H/C4 protein ratio. Rheumatoid factor (RF) levels were also measured. A significant reduction in CH50 and specific C4 activity in HCV patients, compared with the healthy controls, was observed before the onset of treatment; the other parameters were not affected and no C1s-C1 inhibitor complexes were detected. At the same time, a significant reduction in specific C4 activity was observed in relapsers compared with sustained responders. These results point to a potential predictive function of C4 specific activity to monitor the response to therapy. Restoration of specific C4 activity at 6 months was better in responders than in non-responders. Complement activation in chronic hepatitis C does not seem to involve the C1 stage of the classical pathway. A negative correlation between specific C4 activity and RF titres suggests a possible involvement of RF in C4 activation, via the lectin pathway. Specific C4 monitoring appears to be a valuable tool for the follow-up of chronic hepatitis C treatment, together with the other conventional investigations.

Hepatitis C virus (HCV) or HCV-low-density lipoprotein (LDL) complexes interact with the LDL receptor (LDLr) and the HCV envelope glycoprotein E2 interacts with CD81 in vitro. However, E2 interactions with LDLr and HCV interactions with CD81 have not been clearly described. Using sucrose gradient-purified low-density particles (1.03 to 1.07 g/cm(3)), intermediate-density particles (1. 12 to 1.18 g/cm(3)), recombinant E2 protein, or control proteins, we assessed binding to MOLT-4 cells, foreskin fibroblasts, or LDLr-deficient foreskin fibroblasts at 4 degrees C by flow cytometry and confocal microscopy. Viral entry was determined by measuring the coentry of alpha-sarcin, a protein synthesis inhibitor. We found that low-density HCV particles, but not intermediate-density HCV or controls bound to MOLT-4 cells and fibroblasts expressing the LDLr. Binding correlated with the extent of cellular LDLr expression and was inhibited by LDL but not by soluble CD81. In contrast, E2 binding was independent of LDLr expression and was inhibited by human soluble CD81 but not mouse soluble CD81 or LDL. Based on confocal microscopy, we found that low-density HCV particles and LDL colocalized on the cell surface. The addition of low-density HCV but not intermediate-density HCV particles to MOLT-4 cells allowed coentry of alpha-sarcin, indicating viral entry. The amount of viral entry also correlated with LDLr expression and was independent of the CD81 expression. Using a solid-phase immunoassay, recombinant E2 protein did not interact with LDL. Our data indicate that E2 binds CD81; however, virus particles utilize LDLr for binding and entry. The specific mechanism by which HCV particles interact with LDL or the LDLr remains unclear.