Anna Linda Zignego

Liver stiffness measurement predicts severe portal hypertension in patients with HCV‐related cirrhosis

Measurement of hepatic venous pressure gradient (HVPG) is a standard method for the assessment of portal pressure and correlates with the occurrence of its complications. Liver stiffness measurement (LSM) has been proposed as a noninvasive technique for the prediction of the complications of cirrhosis. In this study, we evaluated the ability of LSM to predict severe portal hypertension compared with that of HVPG in 61 consecutive patients with HCV‐related chronic liver disease. A strong relationship between LSM and HVPG measurements was found in the overall population (r = 0.81, P < 0.0001). However, although the correlation was excellent for HVPG values less than 10 or 12 mm Hg (r = 0.81, P = 0.0003 and r = 0.91, P < 0.0001, respectively), linear regression analysis was not optimal for HVPG values ≥10 mm Hg (r2 = 0.35, P < 0.0001) or ≥12 mm Hg (r2 = 0.17, P = 0.02). The AUROC for the prediction of HVPG ≥10 and ≥12 mm Hg were 0.99 and 0.92, respectively and at LSM cutoff values of 13.6 kPa and 17.6 kPa, sensitivity was 97% and 94%, respectively. In patients with cirrhosis, LSM positively correlated with the presence of esophageal varices (P = 0.002), although no correlation between LSM and esophageal varices size was detected. The area under the ROC for the prediction of EV was 0.76 and at a LSM cutoff value of 17.6 kPa sensitivity was 90%. Conclusion: LSM represents a non‐invasive tool for the identification of chronic liver disease patients with clinically significant or severe portal hypertension and could be employed for screening patients to be subjected to standard investigations including upper GI endoscopy and hemodynamic studies. (HEPATOLOGY 2007;45:1290–1297.)

Infection of peripheral mononuclear blood cells by hepatitis C virus

We investigated the infection of peripheral blood mononuclear cells (PBMNC) by hepatitis C virus (HCV) in 5 patients with HCV-related chronic hepatitis. The presence of HCV-RNA-positive and -negative strands was tested with the polymerase chain reaction (PCR) method. In all subjects, HCV-RNA was shown in PBMNC. In 3 cases, HCV-RNA was shown in the T- and B-cell populations, with viral RNA also present in the monocyte-macrophage fraction of two of these. HCV-RNA-negative stranded molecules, indicative of the viral multiplication, were significantly increased in cells maintained in cultures with PHA/PMA stimulation. The results indicate that HCV infect blood mononuclear cells, thus suggesting that this cellular tropism may play a role in HCV infection.

Hepatitis C virus infection in patients with non-Hodgkin's lymphoma

Summary. Hepatitis C virus (HCV), which is both a hepatotropic and a lymphotropic virus, has been proposed as a possible causative agent of mixed cryoglobulinaemia. This ‘benign’ lymphoproliferative disorder can switch over to a malignant B‐cell non‐Hodgkins lymphoma (NHL). Therefore HCV infection has been investigated in a series of 50 unselected Italian patients with B‐cell NHL. Antibodies against HCV were found in 30% of NHL and HCV viraemia in 32% of cases. HCV‐related markers were detected in 34% (17/50) of our NHL patients; this prevalence is particularly significant when compared with HCV seropositivity in Hodgkins lymphoma (3%) and healthy controls (1.3%).

Different cytokine profiles of intraphepatic T cells in chronic hepatitis B and hepatitis C virus infections

BACKGROUND & AIMS The cytokine pattern secreted by T cells at the site of viral replication may influence the final outcome of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. The aim of this study was to assess whether a cytokine imbalance oriented toward T helper (Th) 1 or Th2-type responses may play a role in chronic hepatitis B or C. METHODS Production of interferon (IFN)-gamma, interleukin (IL)-4, and IL-5 by wide series of T-cell clones derived from the liver of 6 patients with chronic hepatitis B (291 clones) and 9 patients with chronic hepatitis C (260 clones) was studied. T-cell clones were generated by limiting dilution from freshly isolated mononuclear cells derived from liver tissue to give a reliable representation of the intrahepatic inflammatory infiltrates. RESULTS The majority of liver-infiltrating T cells in chronic hepatitis C were Th1 cells able to secrete IFN-gamma but unable to secrete IL-4 or IL-5, whereas in hepatitis B, most CD4+ and CD8+ liver T cells were ThO-like cells able to produce not only IFN-gamma but also IL-4 and IL-5. CONCLUSIONS The different cytokine profiles of T cells within the liver in chronic HBV and HCV infections illustrate a different behavior of the local immune response in these two infections that may have pathogenetic implications.

A randomized controlled trial of rituximab for the treatment of severe cryoglobulinemic vasculitis

OBJECTIVE To conduct a long-term, prospective, randomized controlled trial evaluating rituximab (RTX) therapy for severe mixed cryoglobulinemia or cryoglobulinemic vasculitis (CV). METHODS Fifty-nine patients with CV and related skin ulcers, active glomerulonephritis, or refractory peripheral neuropathy were enrolled. In CV patients who also had hepatitis C virus (HCV) infection, treatment of the HCV infection with antiviral agents had previously failed or was not indicated. Patients were randomized to the non-RTX group (to receive conventional treatment, consisting of 1 of the following 3: glucocorticoids; azathioprine or cyclophosphamide; or plasmapheresis) or the RTX group (to receive 2 infusions of 1 gm each, with a lowering of the glucocorticoid dosage when possible, and with a second course of RTX at relapse). Patients in the non-RTX group who did not respond to treatment could be switched to the RTX group. Study duration was 24 months. RESULTS Survival of treatment at 12 months (i.e., the proportion of patients who continued taking their initial therapy), the primary end point, was statistically higher in the RTX group (64.3% versus 3.5% [P < 0.0001]), as well as at 3 months (92.9% versus 13.8% [P < 0.0001]), 6 months (71.4% versus 3.5% [P < 0.0001]), and 24 months (60.7% versus 3.5% [P < 0.0001]). The Birmingham Vasculitis Activity Score decreased only after treatment with RTX (from a mean ± SD of 11.9 ± 5.4 at baseline to 7.1 ± 5.7 at month 2; P < 0.001) up to month 24 (4.4 ± 4.6; P < 0.0001). RTX appeared to be superior therapy for all 3 target organ manifestations, and it was as effective as conventional therapy. The median duration of response to RTX was 18 months. Overall, RTX treatment was well tolerated. CONCLUSION RTX monotherapy represents a very good option for severe CV and can be maintained over the long term in most patients.

Prevalence of bcl-2 Rearrangement in Patients with Hepatitis C Virus–Related Mixed Cryoglobulinemia with or without B-Cell Lymphomas

Context Rearrangement of bcl-2 has an antiapoptotic effect and has been implicated as a potential cause of benign lymphoproliferation (causing mixed cryoglobulinemia) and B-cell lymphoma. Mixed cryoglobulinemia is strongly associated with hepatitis C virus (HCV) infection. Contribution In patients with HCV-associated chronic liver disease, bcl-2 rearrangement occurred significantly more often in patients with chronic HCV infection and mixed cryoglobulinemia than in HCV-infected patients without mixed cryoglobulinemia; it also occurred in three of four patients with B-cell lymphoma. Transient suppression of HCV in two patients was associated with remission of clinical manifestations of mixed cryoglobulinemia. Implications Viral induction of gene sequence translocations may help explain some benign and malignant lymphoproliferative disorders. The Editors Mixed cryoglobulinemia is a distinct syndrome clinically characterized by purpura; weakness; arthralgia; and such conditions as membranoproliferative glomerulonephritis, peripheral neuropathy, skin ulcers, and diffuse vasculitis (1, 2). Cryoprecipitable immune complexes, specifically mixed (IgG-IgM) cryoglobulins, are the serologic hallmark of the disease. Immunoglobulin Gs are the autoantigens, and IgMs with rheumatoid factor activity are the autoantibodies. Mixed cryoglobulinemia is classified as type II or type III according to the presence of polyclonal or monoclonal IgMs (3, 4). Because expansion of rheumatoid factorproducing B cells is the underlying disorder of mixed cryoglobulinemia, this condition is considered a benign B-cell lymphoproliferative disease. Type II and III mixed cryoglobulinemia are similar in terms of organ involvement and clinical course, except that type II disease may evolve into cancer. Type II mixed cryoglobulinemia is often observed in conjunction with bone marrow findings consistent with indolent B-cell lymphoma (5-9) and evolves to frank B-cell malignancy in about 10% of cases (10). A strong association between mixed cryoglobulinemia and infection with hepatitis C virus (HCV), a hepatotropic and lymphotropic virus (10, 11), has been shown. A pathogenetic role of chronic infection with HCV in mixed cryoglobulinemia has been suggested. The mechanisms involved in benign lymphoproliferation of mixed cryoglobulinemia and its evolution to lymphoma remain unknown. However, rearrangement of the antiapoptotic B-cell lymphoma/leukemia 2 (bcl-2) genethe t(14;18) translocationis suggested to play a role in the pathogenesis of HCV-associated mixed cryoglobulinemia (12, 13). The t(14;18) translocation, the most frequent genetic aberration in human lymphoma (14, 15), may be favored by sustained, strong antigenic stimulation (16-18). As a result of bcl-2 rearrangement, the bcl-2 gene on chromosome 18q21 is coupled with the immunoglobulin heavy chain gene (IgH) on chromosome 14q32 by a process frequently involving IgH joining segments (JH) (Figure 1, top). At the junction of the two genes, insertions of variable lengths (N segments) due to random addition of nongermline nucleotides result in a DNA pattern that is clone specific (19, 20). As a consequence of this rearrangement, bcl-2 is activated and B cells bearing the t(14;18) translocation express inappropriately elevated levels of the Bcl-2 protein. Figure 1. Schematic representation of the t(14; 18) translocation and its effects on B cells. Top. bcl-2 Bottom. Bcl-2 is a member of a larger family. Family members can interact with each other in a complex manner; some act to promote and others to inhibit apoptosis (14). The Bcl-2 protein protects cells from apoptosis, whereas its homologue, Bax, kills cells (21). Thus, the ratio of Bcl-2 to Bax is a determinant of susceptibility to apoptosis (14) (Figure 1, bottom). Strong expression of Bcl-2 protein has been observed in lymphoid infiltrates in liver and bone marrow specimens of patients with mixed cryoglobulinemia (22). In a previous study, the prevalence of bcl-2 rearrangement in peripheral blood mononuclear cells was significantly higher in patients with chronic HCV infection than in healthy persons or those without HCV infection but with chronic liver diseases or systemic autoimmune disorders (13). Of note, the prevalence of bcl-2 rearrangement was particularly high in patients with HCV-associated type II mixed cryoglobulinemia. We sought to evaluate the prevalence of bcl-2 rearrangement in peripheral blood cells of patients with mixed cryoglobulinemia, to confirm that results are patient specific by sequencing studies, to analyze Bcl-2 expression and the ratio of Bcl-2 to Bax in these patients, and to observe the effect of antiviral therapy. Methods Patients We enrolled 37 patients (12 men and 25 women; mean age SD, 64 9 years) with HCV infection and mixed cryoglobulinemia who were consecutively referred to the outpatient clinic of the Department of Internal Medicine, University of Florence School of Medicine, a tertiary hepatology center, and the rheumatologic section of the Department of Internal Medicine, University of Pisa School of Medicine, from January 1999 to May 2000. These patients were compared with 101 consecutively recruited patients (62 men and 39 women; mean age, 51 11 years) who had HCV-related chronic liver diseases but not mixed cryoglobulinemia or another lymphoproliferative disease. Hepatitis C virus infection was established by detection of circulating anti-HCV antibodies (EIA-2 and RIBA-2, Ortho Diagnostic Systems, Raritan, New Jersey) and HCV RNA (nested polymerase chain reaction [PCR] for HCV) (10, 13, 23). Essential mixed cryoglobulinemia was diagnosed according to published criteria (10, 13). Serum cryoglobulins, complement fraction levels, rheumatoid factor, and autoantibodies were routinely measured and characterized in all patients as described elsewhere (10, 13, 23). Diagnosis of liver disease was based on results of liver biopsy. Lymphomas were diagnosed by an independent pathologist and classified according to the revised European-American classification of lymphoid neoplasms (24). No patient tested positive for hepatitis B surface antigen, IgM anti-HBc, hepatitis B virus DNA, IgM anti-delta, antiEpsteinBarr virus, anti-cytomegalovirus, antiherpes simplex virus, or anti-HIV. No patient had a history of alcohol abuse or previous antiviral or immunosuppressive treatment. All patients gave informed consent to participate in the study, which was performed in accordance with the principles of the Declaration of Helsinki, and the study was approved by the local ethics committee. Detection of the t(14; 18) Translocation The t(14; 18) translocation in peripheral blood mononuclear cells was detected on total DNA by using nested PCR (major breakpoint region), as described elsewhere (13). Nested PCR is a variant of PCR; after an initial series of amplification cycles, templates are again amplified by using a second set of primers internal to the first ones. The resulting reaction is very specific and sensitive owing to specific binding to the target sequences of four instead of two specific primers. The limit of sensitivity was one rearranged cell in 105 to 106 normal cells. Amplification products were analyzed by both ethidium bromide staining and hybridization with a specific digoxigenin-labeled probe (Southern blot analysis). Each sample was analyzed at least twice, and all samples that tested negative on PCR were analyzed at least four times. Different cell samples that were obtained at the same time (synchronous) or at different times (metachronous) were also analyzed when possible. Approximately 2.5 105 mononuclear cells were tested in each reaction, corresponding to about 1 g of DNA. Positive and negative control samples were included in each experiment (13). To avoid false-positive results caused by carryover of PCR product, precautions were taken, as described elsewhere (10, 13). To ensure DNA amplificability, PCR was also performed by using primers for the human HLA gene (exon 2 of HLA-DRB gene), as previously reported (13). Finally, bcl-2/JH junction sequence was determined in part by cycle sequencing and solid-phase sequencing techniques (13, 25) and in part by automated sequencing (Abi Prism, Perkin Elmer, Norwalk, Connecticut). Measurement of Bcl-2 and Bax Proteins Bcl-2 and Bax proteins were measured as described elsewhere (13) on freshly isolated peripheral blood mononuclear cells and, when possible (9 patients), in separated cell subgroups (T cells, B cells, and monocytes and macrophages). Bcl-2 was detected by using monoclonal mouse anti-human Bcl-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, California), and Bax was detected by using polyclonal rabbit anti-human Bax (Upstate Biotechnology, Inc., Lake Placid, New York). The CD2+ T cells, CD19+ B cells, and CD14+monocytes and macrophages from peripheral blood were separated by immunomagnetic isolation using Dynabeads M450 Pan-T, M-450 Pan-B, and M-450 CD14+, respectively (Dynal A.S., Oslo, Norway), according to the manufacturers instructions. Statistical Analysis Data are expressed as the mean SD. Data were analyzed by performing the Fisher exact test, using True Epistat 4.0 statistical software (Epistat Service, Richardson, Texas). A P value less than 0.05 was considered significant. Role of the Funding Sources The funding sources had no role in the analysis, reporting, or interpretation of the data or in the decision to submit the report for publication. Results The Table shows the clinical, epidemiologic, and pathologic characteristics of patients with HCV-related mixed cryoglobulinemia. The mean duration of mixed cryoglobulinemia syndrome was 9.2 5.2 years. Most of these patients (91%) had chronic liver diseases. Liver biopsy showed chronic hepatitis in 27 patients (72.9%) and cirrhosis in 7 patients (18.9%); of the latter patients, 1 also had superimposed hepatocellular carcinoma. Liver biopsy was not performed in the remaining 3 patients becaus

Hepatitis C Virus Genotype Analysis in Patients with Type II Mixed Cryoglobulinemia

Hepatitis C virus (HCV) infection has been related to different autoimmune-lymphoproliferative diseases such as autoimmune hepatitis [1, 2] and mixed cryoglobulinemia [3, 4]. The latter condition is associated with HCV infection in almost 90% of cases and is characterized by symptoms of systemic vasculitis secondary to deposition of coldprecipitable immune complexes. The remote pathogenesis of mixed cryoglobulinemia is considered to be a B-cell lymphoproliferation, which in many patients can be complicated by malignant lymphoma [5, 6]. Because of the variability of the HCV genome, one might speculate that particular viral variants are responsible for mixed cryoglobulinemia. Thus, we assessed the prevalence of different genotypes in HCV-positive cryoglobulinemic patients and in patients with chronic HCV infection who did not have cryoglobulinemia. Methods From March 1994 to September 1994, we recruited (at ambulatory visits) 29 consecutive HCV-positive (anti-HCV antibody-positive and HCV RNA-positive) patients with type II (IgM ) mixed cryoglobulinemia (9 men, 20 women; mean age SD, 60 7.5 years; age range, 46 to 72 years) and 61 patients with chronic HCV infection who did not have mixed cryoglobulinemia (control group). All patients studied were Italian-born, were heterosexual, and had no history of blood transfusion or drug or alcohol abuse. Cryoglobulinemic and control patients were followed at the rheumatology and hepatology units of the University of Pisa and University of Florence. The mean (SD) duration of follow-up was 8.4 5.5 years (range, 1 to 25 years) for cryoglobulinemic patients and 6.8 4.5 years (range, 2 to 14 years) for controls. A diagnosis of mixed cryoglobulinemia was made if a patient had the typical syndrome (purpura, arthralgias, weakness, and circulating mixed cryoglobulins) and if other well-known systemic disorders could be ruled out. Eight cryoglobulinemic patients developed B-cell non-Hodgkin lymphoma 4.3 2.7 years (range, 1.5 to 8 years) after diagnosis. After informed consent was obtained, percutaneous liver and renal biopsies were done as previously described [4, 7]. Cryocrit determinations were done and cryoglobulin composition was evaluated as previously described [4, 8]. Antinuclear, anti-smooth muscle, anti-liver-kidney microsomal 1, anti-soluble liver antigen, and antimitochondrial autoantibodies were assayed by current techniques [9]. A titer greater than 1:40 was considered positive. Anti-extractable nuclear antigen antibody determinations were done using the method of Bunn and colleagues [10]. Serum samples and aliquots of peripheral blood mononuclear cells with the last washing liquid (phosphate-buffered saline) for HCV polymerase chain reaction (PCR) analysis were collected as previously described [11, 12]. In addition, to ascertain the presence of a latent HCV infection, peripheral blood mononuclear cell samples were cultured for 72 hours in the presence of mitogens (phytohemagglutinin-phorbol myristate acetate) as previously described [11, 12]. Different samples were tested by one-tube nested reverse transcriptase PCR with primers derived from the 5 noncoding region [13]. Several precautions were taken to prevent false-positive results [14], including the incorporation of deoxyuridane-triphosphate instead of deoxythymidine-triphosphate during amplification steps followed by incubation of PCR mixtures for 3 minutes at 50 C in the presence of uracil-N-glycosilase (UNG; Perkin Elmer Cetus, Norwalk, Connecticut). In nine unselected patients with mixed cryoglobulinemia, aliquots of peripheral blood mononuclear cells were also available for HCV genotyping. Hepatitis C virus genotyping was done using two different methods, both based on amplification by PCR. The first technique used type-specific primers localized in the core region, as described by Okamoto and colleagues [15], with the difference that PCR was done without mixing genotype-specific antisense primers. Moreover, for the detection of genotype III, we used a new primer that, in a previous study, made it possible to classify most previously unclassified HCV isolates as genotype 2a/III [16]: This primer was CRIIIa antisense 5-TTCCCCAGGAYT TGCCAGTGG-3 (Y equals C or T). The second one employed biotinyled, universal primers localized in the 5 noncoding region of HCV RNA; amplification products were then hybridized to genotype-specific probes (Line Probe Assay, LiPA, Innogenetics, Brussels, Belgium). Statistical analysis was done using the chi-square test and the Fisher exact test whenever the z approximation was inadequate. Results Table 1 shows the values for the main clinicoepidemiologic and laboratory variables in patients with mixed cryoglobulinemia. The following complications of mixed cryoglobulinemia were recorded: peripheral neuropathy in 15 of 29 patients (52%); mild sicca syndrome in 11 of 28 (39%); glomerulonephritis in 4 of 29 (13%); Raynaud phenomenon in 1 of 28 (4%); and skin ulcers in 3 of 28 (11%). One or more serum autoantibodies were detected in 8 of 28 (29%) patients with mixed cryoglobulinemia and in 19 of 61 (31%) controls. Table 1. Clinico-epidemiologic Data and Laboratory Findings in 29 Hepatitis C Virus RNA-Positive Patients with Mixed Cryoglobulinemia* Hepatitis C virus RNA sequences were shown in uncultured peripheral blood mononuclear cells from 23 of 29 (75%) patients with mixed cryoglobulinemia and in cultured cells from 3 other patients (total, 90%) (Table 1). In the control group, viral sequences were detected in uncultured or mitogenstimulated peripheral blood mononuclear cells from 46 of 61 (75%) and 49 of 61 persons (total, 80%), respectively (Table 1). Among the 29 patients with mixed cryoglobulinemia, serum specimens showed a single infection with type 1a/I in 1 patient (3 %), with type 1b/II in 14 patients (48%), and with type 2a/III in 12 patients (41%). Two patients (6.6%) had mixed infection (1a/I plus 1b/II and 1b/II plus 2a/III, respectively) (Table 1). Among the 61 controls, genotypes 1a/I, 1b/II, 2a/III, 3a/V, and 4a were observed in 7 (11%), 37 (61%), 9 (15%), 4 (7%), and 1 (1%) patient, respectively, whereas mixed infection (1a/I plus 1b/II; 1b/II plus 2a/III; 1b/II plus 3a/V) was observed in 3 (5%) patients. When HCV genotypes detected in peripheral blood mononuclear cells were also considered, type 2a/III was found in 15 of the 29 (52%) patients with mixed cryoglobulinemia and in most autoantibody-positive patients (6 of 8; 75%) (Table 1). The prevalence of 2a/III genotype was significantly higher in patients with mixed cryoglobulinemia (12 of 29; 41%) than in controls (9 of 61; 15%), a difference of 27 percentage points (95% CI, 6.6% to 46.6%; P = 0.009). No other significant differences were observed between the two groups. Sixteen of the 29 patients with mixed cryoglobulinemia had chronic aminotransferase elevations. Analysis of serum samples showed that 12 of these patients (75%) were infected with HCV genotype 1b/II and that 4 (25%) were infected with HCV genotype 2a/III (Table 1). Of the remaining 13 patients who showed no clinical evidence of liver damage, 8 (61%) had infection with genotype 2a/III, 3 (23%) had infection with genotype 1b/II, 1 (7%) had infection with genotype 1a/I, and 1 had coinfection with types 1b/II and 2a/III. Liver biopsy, done in 14 patients, showed chronic hepatitis in 13 patients and liver cirrhosis in 1 patient; 2 patients with chronic hepatitis and 1 patient with cirrhosis had persistently normal aminotransferase levels (Table 1). Among the 61 controls, 27 (44%) had chronic hepatitis, 21 (34%) had liver cirrhosis, and 13 (21%) had hepatocellular carcinoma; none had normal aminotransferase values. Discussion In our study, HCV genotype 2a/III had a significantly higher prevalence in HCV-positive patients with mixed cryoglobulinemia than in patients with chronic hepatitis who did not have cryoglobulinemia. Among cryoglobulinemic patients, this genotype was more frequent in those without a symptomatic liver disease or with circulating autoantibodies. Recently, several reports have suggested different clinical outcomes for the HCV genotypes. Type 1b/II infection, for example, has been associated with a more severe liver disease and a lower response to interferon treatment, whereas type 2a/III infection has been considered relatively benign [17-19]. This hypothesis is consistent with the observation that genotype 2a/III is more prevalent in cryoglobulinemic patients without symptomatic liver disease than in those with chronic hepatitis. On the other hand, the higher prevalence of genotype 2a/III in patients with mixed cryoglobulinemia than in controls, especially in cryoglobulinemic patients with circulating autoantibodies, suggests that type 2a/III might be involved in the pathogenesis of autoimmune-lymphoproliferative disorders. The recent observation that type 2a/III is particularly frequent in Italian patients with anti-liver-kidney microsomal 1 autoantibody-positive type 2 autoimmune hepatitis further supports the possibility of a peculiar pathogenetic role for this genotype [20]. A recent study [8] showed that patients with mixed cryoglobulinemia have a high prevalence (81%) of HCV infection in peripheral blood mononuclear cells, suggesting that HCV lymphotropism may play a key role in determining the lymphoproliferative disorder underlying the disease. Our study confirms these data and also shows the frequent infection of lymphatic cells in HCV-positive patients with chronic hepatitis who do not have cryoglobulinemia. We can thus hypothesize that different viral, genetic, or environmental factors, in addition to the infection of lymphatic cells, may be involved in the pathogenesis of this disorder. The exact role of HCV variants, namely 2a/III, which are possibly related to different host immune reactivity or to a greater lymphotropism, should be clarified through deeper virologic analysis, including examination of lymph-node and bone

Extrahepatic manifestations of chronic hepatitis C virus infection

Hepatitis C virus (HCV) infected patients are known to be at risk of developing liver complications i.e. cirrhosis and liver cancer. However, the risks of morbidity and mortality are underestimated because they do not take into account non-liver consequences of chronic hepatitis C virus infection. Numerous extrahepatic manifestations have been reported in up to 74% of patients, from perceived to disabling conditions. The majority of data concern hepatitis C virus-related autoimmune and/or lymphoproliferative disorders, from mixed cryoglobulinaemia vasculitis to frank lymphomas. More recently, other hepatitis C virus-associated disorders have been reported including cardiovascular, renal, metabolic, and central nervous system diseases. This review aims to outline most of the extrahepatic manifestations that are currently being investigated, including some of autoimmune and/or lymphoproliferative nature, and others in which the role of immune mechanisms appears less clear. Beyond the liver, hepatitis C virus chronic infection should be analyzed as a multifaceted systemic disease leading to heavy direct and indirect costs. The accurate consideration of extrahepatic consequences of such a systemic infection significantly increases the weight of its pathological burden. The need for effective viral eradication measures is underlined.

Persistent Hepatitis B Virus Infection Of Mononuclear Blood Cells Without Concomitant Liver Infection: The Liver Transplantation Model

We have investigated the recurrence of Hepatitis B virus (HBV) in 30 patients treated by orthotopic liver transplantation and given high doses of anti-HBs immunoglobulin. The polymerase chain reaction (PCR) assay showed no evidence of HBV DNA sequences in the liver of 23/24 patients who remained serum HBsAg-negative during a mean follow-up of 13 months (2-24 months) after OLT. However, the liver scored positive in all the 6 individuals in whom HBsAG reappeared. The PCR assay identified HBV DNA sequences in the peripheral blood mononuclear cells of 7 of 11 subjects who were serum HBsAG-negative and liver HBV DNA-negative by PCR. Therefore, this application of the sensitive PCR assay demonstrates persistent infection of PBMC in the absence of liver HBV--thus OLT provides a model for studying the interaction between HBV, PBMC, and the liver.

Extrahepatic manifestations of HCV infection: facts and controversies

HCV Infection and Lymphoproliferative Disorders Mixed cryoglobulinaemia The most documented extrahepatic manifestation of HCV infection is essential mixed cryoglobulinaemia (EMC). This topic has been the object of a previous, exhaustive, review by Lunel & Musset (1). Briefly, EMC is characterised by the presence of temperaturesensitive protein complexes: in type II EMC, cryoglobulins are composed of a monoclonal rheumatoid factor (RF) (usually IgMK) against polyclonal IgG. In type III EMC, all components are polyclonal. Since monoor polyclonal B lymphocyte expansion is responsible for cryoglobulin production, EMC may be considered a lymphoproliferative disorder (LPD). Due to the fact that EMC frequently coexists with bone marrow aspects of B-cell non-Hodgkin’s lymphoma (NHL) and may evolve, in about 5–8% of cases, into a frank Bcell malignancy, it may be considered a ‘‘borderline’’ (benign/malignant) LPD. Evidence in favour of a pathogenetic link between EMC and HCV derives from a series of data including, first, the very high prevalence of HCV markers in EMC patients (1–8) with detection of HCV sequences in peripheral and bone marrow mononuclear cells in most patients, also in the absence of viraemia (9). The characteristic occurrence of EMC in patients with a longer history of HCV infection and/or elevated age suggests that EMC development depends on the length of infection (1,10,11). In the single patient, however, inherited or acquired factors predisposing to LPDs may accelerate the occurrence of cryoglobulinaemia and worsen corresponding clinical manifestations, as also suggested by observations made in patients receiving a liver graft for HCV-related cirrhosis (12).