Monica Monti

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%).

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

Relevance of inapparent coinfection by hepatitis B virus in alpha interferon-treated patients with hepatitis C virus chronic hepatitis.

The aim of the study was to investigate whether an “inapparent” coinfection by hepatitis B virus (HBV) in anti‐HCV‐positive chronic liver disease patients may influence interferon (IFN) response. Fourteen anti‐HCV‐positive, hepatitis B surface antigen (HBsAg)‐negative but serum HBV‐DNA‐positive patients and 111 anti‐HCV‐positive, HBsAg‐negative, and HBV‐DNA‐negative patients with chronic hepatitis were treated with 3 MU of recombinant α‐2a IFN 3/week for 1.2 months. Serum HBV‐DNA and HCV‐RNA were determined before treatment, after 6–12 months, and at the time of alanine aminotransferase (ALT) flare‐up by HBV polymerase chain reaction (PCR) and HCV PCR, respectively. IgM anti‐HBc were tested using the IMx Core‐M assay (Abbott Laboratories, North Chigago, IL). By the end of treatment, ALT values had become normal in 4/14 HBV‐DNA‐positive patients (28%), but all “responders” (4/4) relapsed. IgM anti‐HBc was detected both before treatment and during ALT elevation in three patients and only during ALT relapse in another three. In the remaining 111 patients, a biochemical response to IFN treatment was observed in 54% and relapse of ALT values in 47%. “Inapparent” HBV/HCV coinfection may be implicated in cases of resistance to IFN. HBV replication and HBV‐related liver damage may persist in patients in whom HCV replication was inhibited by current doses of IFN, as suggested also by the presence of IgM anti‐HBc in some cases. Further studies will show the effect of different treatment schedules. HBV‐DNA and/or IgM anti‐HBc detection with very sensitive methods may be important both as a prognostic factor and as a tool for better understanding of intervirus relationships and mechanisms involved in multiple hepatitis virus infections. J. Med. Virol. 51:313–318, 1997.

Long-term effect of HCV eradication in patients with mixed cryoglobulinemia: A prospective, controlled, open-label, cohort study

Limited data are available about the efficacy of antiviral treatment in hepatitis C virus (HCV)–associated mixed cryoglobulinemia (MC), especially concerning the long‐term effects of HCV eradication. The aim of this study was to evaluate the influence of MC on the virological response and the long‐term effects of viral eradication on MC. We prospectively enrolled 424 HCV+ patients belonging to the following groups: MC syndrome (MCS)‐HCV (121 patients with symptomatic MC), MC‐HCV (132 patients with asymptomatic MC), and HCV (158 patients without MC). Pegylated interferon plus ribavirin treatment was administered according to standard protocols. Posttreatment follow‐up ranged from 35 to 124 months (mean 92.5 months). A significant difference was observed in the rate of sustained virological response between the HCV group and both the MC‐HCV (P = 0.009) and MC‐HCV+MCS‐HCV (P = 0.014) groups. Multivariate logistic regression analysis identified cryoglobulinemia as an independent prognostic factor of nonresponse. The clinical–immunological response in MCS‐HCV correlated with the virological one. All patients with sustained virological response also experienced a sustained clinical response, either complete or partial. In the majority of sustained virological response patients all MCS symptoms persistently disappeared (36 patients, 57%); in only two (3%) did definite MCS persist. All virological nonresponders were also clinical nonresponders, in spite of a transient improvement in some cases. No evolution to lymphoma was observed. For the first time we have evaluated both the effects of interferon‐based therapy on HCV patients with and without MC and with and without symptoms, as well as the long‐term effects of viral eradication on MC. Conclusion: MC is a negative prognostic factor of virological response. Clearance of HCV led to persistent resolution or improvement of MCS, strongly suggesting the need for a next generation of highly effective antiviral drugs. (Hepatology 2015;61:1145‐1153)

Prospective study of guideline-tailored therapy with direct-acting antivirals for hepatitis C virus-associated mixed cryoglobulinemia.

Hepatitis C virus (HCV)‐associated mixed cryoglobulinemia (MC) vasculitis commonly regresses upon virus eradication, but conventional therapy with pegylated interferon and ribavirin yields approximately 40% sustained virologic responses (SVR). We prospectively evaluated the efficacy and safety of sofosbuvir‐based direct‐acting antiviral therapy, individually tailored according to the latest guidelines, in a cohort of 44 consecutive patients with HCV‐associated MC. In two patients MC had evolved into an indolent lymphoma with monoclonal B‐cell lymphocytosis. All patients had negative HCV viremia at week 12 (SVR12) and at week 24 (SVR24) posttreatment, at which time all had a clinical response of vasculitis. The mean (±standard deviation) Birmingham Vasculitis Activity Score decreased from 5.41 (±3.53) at baseline to 2.35 (±2.25) (P < 0.001) at week 4 on treatment to 1.39 (±1.48) (P < 0.001) at SVR12 and to 1.27 (±1.68) (P < 0.001) at SVR24. The mean cryocrit value fell from 7.2 (±15.4)% at baseline to 2.9 (±7.4)% (P < 0.01) at SVR12 and to 1.8 (±5.1)% (P < 0.001) at SVR24. Intriguingly, in the 2 patients with MC and lymphoma there was a partial clinical response of vasculitis and ∼50% decrease of cryocrit, although none experienced a significant decrease of monoclonal B‐cell lymphocytosis. Adverse events occurred in 59% of patients and were generally mild, with the exception of 1 patient with ribavirin‐related anemia requiring blood transfusion. Conclusion: Interferon‐free, guideline‐tailored therapy with direct‐acting antivirals is highly effective and safe for HCV‐associated MC patients; the overall 100% rate of clinical response of vasculitis, on an intention‐to‐treat basis, opens the perspective for curing the large majority of these so far difficult‐to‐treat patients. (Hepatology 2016;64:1473‐1482)

Low serum tryptophan levels, reduced macrophage IDO activity and high frequency of psychopathology in HCV patients.

Summary.  Indoleamine 2,3‐dioxygenase (IDO), a key enzyme of tryptophan (TRP) metabolism, is induced in various tissues of patients with bacterial and viral infection or with neoplastic diseases. This induction is considered the main cause of the decreased serum TRP levels, the reduced brain serotonin synthesis and the occurrence of psychopathological disorders often detected in patients with chronic infections or different forms of cancer. We studied 89 subjects including: (a) 39 patients with chronic hepatitis C virus (HCV) infection and mild liver damage (b) 40 healthy controls, and (c) 10 patients with chronic hepatitis B virus (HBV) infection. We measured serum TRP and kynurenine levels and IDO activity in macrophages. Furthermore, each patient had an accurate psychopathological evaluation. HCV‐infected patients had lower (−28%) serum TRP concentrations than healthy volunteers or HBV‐infected patients with comparable liver damage. Depression and anxiety symptoms were particularly common in HCV patients. Unexpectedly, serum kynurenine levels and IDO activity in cultured macrophages (under both basal or stimulated conditions) were lower in HCV patients than in controls. Our study shows that HCV patients have reduced serum TRP levels and confirms that they frequently suffer from anxiety and depression‐related symptoms. The reduced IDO activity found in the macrophages of these patients suggest that HCV infection may hamper macrophage functions.

A phase II, single-arm multicenter study of low-dose rituximab for refractory mixed cryoglobulinemia secondary to hepatitis C virus infection.

Eradication of hepatitis C virus (HCV) by antiviral therapy is the treatment of choice for mixed cryoglobulinemia secondary to this infection, but many patients fail to achieve sustained viral responses and need second-line treatments. Several studies have demonstrated that the infusion of the anti-CD20 monoclonal antibody rituximab is highly effective for refractory mixed cryoglobulinemia, with a clinical response in approximately 80% of patients, although the relapse rate is high. Virtually all published studies employed a rituximab dosage of 375mg/m(2) given four times, a schedule used for treating non-Hodgkins lymphomas. Based on a prior pilot study, we designed a phase II single-arm two-stage study (EUDRACT n. 2008-000086-38) to evaluate the efficacy of a lower dosage of rituximab, 250mg/m(2) given twice, for refractory mixed cryoglobulinemia. We present here the preliminary results in the first 27 patients enrolled. The overall response rate in 24 evaluable patients was 79%, and the mean time to relapse was 6.5months, similar to the 6.7months reported in studies with high-dose rituximab. Side effects were comparable to those seen in patients treated with high-dose. Increase of HCV viral load, reported in some high-dose studies, was not observed in our patients. Low-dose rituximab may provide a more cost/effective and possibly safer alternative for treating refractory HCV-associated mixed cryoglobulinemia.

Prevalence of mixed infection by different hepatitis C virus genotypes in patients with hepatitis C virus–related chronic liver disease☆☆☆★

Multiple infection by different hepatitis C virus (HCV) genotypes may be of great clinico-pathologic interest. In this study we determined the effective prevalence of coinfections by two or more HCV genotypes in 213 subjects with HCV-positive chronic hepatitis by using genotype-specific polymerase chain reaction (PCR), genotype-specific probe hybridization, and direct sequencing. The most prevalent genotype was HCV-1b (54%). HCV-2 (a/c) was also prevalent (27%), and types 1a and 3a were found in 5% and 3% of patients, respectively. A mixed infection was detected in 23 patients (10.8%): 4 out of 23 were coinfected by types 1a + 1b, while the remaining 19 patients had a b + 2 (a/c) mixed infection. Further analysis based on restriction fragment length polymorphism (RFLP) on type-specific PCR products was used to verify genotyping results. Only four coinfections (1a + 1b in 2 patients and 1b + 2 (a/c) in the remaining 2 patients, respectively) were confirmed by enzyme cleavage. All patients with true coinfection had long-lasting infection and liver cirrhosis. Both true and false mixed infections resulting from RFLP analysis were confirmed by direct sequencing of type-specific amplification products. We also determined a recurrent C/T transversion at position 618 in all sequenced samples. In 4 cases another point mutation (G/A at position 626) was found, reducing the number of mismatches between HCV-2 and HCV-1b from 4 to 3 (or 2). Interestingly, all HCV-2 isolates sequenced showed the highest degree of nucleotide homology with HCV-2 subtype c, confirming the relatively high prevalence of this subtype in Italy. In conclusion, we showed the possibility of multiple infection by different HCV types in the general population of chronically infected patients without particular risk factors, even if in a low percentage of cases. Further studies are needed to assess the clinical relevance of chronic HCV infection with multiple genotypes.