F. Lombardini

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%).

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

Sonographic analysis of the ankle in patients with psoriatic arthritis.

Foot involvement is very frequent in patients affected by psoriatic arthritis (PsA). However, evaluation of the painful foot can be problematic, because it is often difficult to distinguish between arthritis, tenosynovitis, and enthesopathy. Plain radiographs can show bone erosion or other features of joint involvement, but give little information about the soft tissues. We therefore studied foot involvement in 31 PsA patients using high resolution sonography, and compared the results with the findings on x-ray and clinical examination. Ultrasound revealed pathological findings in a large proportion of the patients, most of whom exhibited no clinical (pain or swelling) or radiological signs of foot involvement at the time of the study. Our data suggest that involvement of the tendons and entheses may be more frequent in PsA patients than has thus far been supposed, even in cases of not particularly aggressive disease, and that clinical evaluation tends to underestimate these manifestations.Foot involvement is very frequent in patients affected by psoriatic arthritis (PsA). However, evaluation of the painful foot can be problematic, because it is often difficult to distinguish between arthritis, tenosynovitis, and enthesopathy. Plain radiographs can show bone erosion or other features of joint involvement, but give little information about the soft tissues. We therefore studied foot involvement in 31 PsA patients using high resolution sonography, and compared the results with the findings on x-ray and clinical examination. Ultrasound revealed pathological findings in a large proportion of the patients, most of whom exhibited no clinical (pain or swelling) or radiological signs of foot involvement at the time of the study. Our data suggest that involvement of the tendons and entheses may be more frequent in PsA patients than has thus far been supposed, even in cases of not particularly aggressive disease, and that clinical evaluation tends to underestimate these manifestations.

Cryoglobulinemic Membranoproliferative Glomerulonephritis Associated with Hepatitis C Virus

A striking association between hepatitis C virus (HCV) and mixed cryoglobulinemia (MC) has been reported by various authors, regardless of the presence of chronic hepatitis. The aim of this study is to evaluate the prevalence of HCV-related markers in cryoglobulinemic membranoproliferative glomerulonephritis (MPGN) which is one of the most severe complications of MC. Antibodies against HCV have been detected by second-generation Chiron ELISA and RIBA in 26/26 (100%) cryoglobulinemic MPGN. In addition, serum HCV RNA, expression of the ongoing viral replication, was present in 7/7 patients by the polymerase chain reaction technique. The high percentage of anti-HCV seropositivity suggests that this virus may play an important role in the pathogenesis of this immunemediated glomerulonephritis.

Hepatitis C virus chronic infection as a common cause of mixed cryoglobulinaemia and autoimmune liver disease

Abstract. Objectives. Mixed cryoglobulinaemia (MC) and autoimmune chronic hepatitis (AI‐CH) are frequently associated with hepatitis C virus (HCV) chronic infection. Because HCV represents a possible common aetiological factor, the aim of the present study is to investigate the clinico‐serological alterations of both MC and AI‐CH and to verify a possible overlap between these disorders.

Effect of alpha-interferon on hepatitis C virus chronic infection in mixed cryoglobulinemia patients

SummarySince a striking association between hepatitis C virus (HCV) infection and mixed cryoglobulinemia has been demonstrated, the aim of this study is to investigate the effect of alpha-interferon (α-IFN) on HCV viraemia and clinico-serological manifestations of 15 patients (ten female and five male, mean [±SD] age 53±7 years). In 14/15 patients pre-study steroid dosage remained unchanged during treatment. α-IFN was administered at a dose of 2×106 IU daily for a month, then every other day for five months. On the whole, a statistically significant improvement of purpura (p<0.001), serum transaminases (p<0.001), and cryocrit (p<0.01) was observed after α-IFN treatment. HCV viraemia was detected by polymerase chain reaction technique in 13/15 patients with mixed cryoglobulinemia and anti-GOR antibodies, expression of HCV-related autoimmunity, were present in 8/15. After α-IFN treatment, HCV RNA levels showed a clear-cut reduction in five persons and disappeared in another, while anti-HCV antibodies (Chiron ELISA and RIBA II) did not change after the six-month period of therapy. These data further support the possible etiopathogenetic role of HCV in patients with mixed cryoglobulinemia and suggest that α-IFN may be regarded as the elective treatment in this disease.ZusammenfassungZwischen der Hepatitis C Virus-Infektion (HCV) und gemischter Kryoglobulinämie wurden bemerkenswerte Beziehungen festgestellt. Bei 15 Patienten mit gemischter Kryoglobulinämie wurde daher die Wirkung von alpha- Interferon (α-IFN) auf die HCV-Virämie und die klinischen und serologischen Parameter der Kryoglobulinämie geprüft (zehn Frauen, fünf Männer, mittleres Alter (±SD) 53±7 Jahre). α-IFN wurde während eines Monats täglich in einer Dosis von 2×106 IU und dann fünf Monate lang jeden zweiten Tag verabreicht. Die Kortikosteroiddosen blieben dabei in 14/15 Fällen unverändert. Nach der α-IFN-Therapie war eine signifikante Besserung der Purpura (p<0,001), der Serumtransaminasen (p<0,001) und des Kryokrit (p<0,01) festzustellen. Bei 13/15 Patienten wurde mittels Polymerase-Kettenreaktion (PCR) eine HCV-Virämie nachgewiesen. Bei 8/15 fanden sich anti-GOR-Antikörper, die als Zeichen der HCV-assoziierten Autoimmunreaktion zu werten sind. Die HCV-RNA-Spiegel waren nach Behandlung mit α-IFN in fünf Fällen deutlich zurückgegangen und in einem Fall verschwunden. Zu einer änderung der anti-HCV Antikörper (Chiron ELISA und RIBA II) kam es während der sechsmonatigen Behandlung mit α-IFN jedoch nicht. Die Ergebnisse unterstützen die Annahme einer ätiopathogenetischen Beziehung zwischen HCV-Infektion und gemischter Kryoglobulinämie. Möglicherweise stellt α-IFN eine gezielte Therapie bei dieser Krankheit dar.

Lymphotropic Virus Infection of Peripheral Blood Mononuclear Cells in B-Cell Non-Hodgkin’s Lymphoma

Some lymphotropic viruses such as Epstein-Barr virus (EBV) and human herpesvirus 6 (HHV-6) have been proposed as causative agents of B cell non-Hodgkins lymphoma (NHL). More recently, the presence of hepatitis C virus (HCV), which is both a hepatotropic and lymphotropic virus, has been reported in one third of B cell NHL patients. The aim of this study was to investigate in a series of B cell NHL the prevalence of three lymphotropic viruses, i.e. EBV, HHV-6 and HCV, in peripheral blood mononuclear cells (PBMC). Eighteen unselected B cell NHL patients (10 men, 8 women; mean age 62 +/- 12 years, range 31-77 years; mean disease duration 1.8 +/- 1.4 years) and 40 age- and sex-matched healthy controls were included in the study. In all cases, an acquired-immunodeficiency-syndrome-related lymphoma was excluded. By means of the polymerase chain reaction technique, EBV DNA, HHV-6 DNA and HCV RNA were detected in PBMC. HCV genomic sequences were significantly more frequent in PBMC of NHL patients than in controls (33 vs. 2.5%; p < 0.01); on the other hand, in the same two groups EBV DNA (39 vs. 60%; p = not significant) and HHV-6 DNA (22 vs. 32%; p = not significant) were present in a comparable percentage of individuals in the same two groups. The infection of PBMC by HCV alone was present in the majority (5 of 6) of HCV-positive NHL. These data support the implication of HCV infection in a statistically significant number of B cell NHL, whereas a possible co-operation between HCV and other well-known lymphotropic viruses seems to be excluded.

Mixed cryoglobulinemia: the role of HCV and organ-specific antibodies

The term ‘cryoglobulin’ was introduced to define a group of proteins which have the common property of forming a reversible precipitate or gel in the cold [1]. Three groups of cryoglobulins have been identified. Type I cryoglobulins are monoclonal, the most common one being immunoglobulin M (IgM), a protein associated with immunoprolifera-tive disorders, such as Waldenstrom’s macroglobulinemia, lymphopro-liferative disorders and multiple myeloma.