Haemochromatosis gene mutations in a clustered Italian population: evidence of high prevalence in people of Celtic ancestry.

Hereditary haemochromatosis is an inherited disorder characterised by an excessive iron absorption from the diet and is associated with several HFE gene mutations. One hypothesis is that these genetic mutations originated in the Celtic populations. The aim of this study is to determine the frequency of HFE gene mutations in a clustered Italian population of Celtic ancestry (Cimbri, Asiago plateau). One hundred and forty-nine consecutive unrelated blood donors (31 females and 118 males) were enrolled in this study. A family investigation was performed in each case to identify the ethnic origin of the individuals. The analysis of HFE gene mutations was performed by PCR amplification followed by digestion with RsaI and DpnII restriction enzymes. At least one HFE gene mutation was identified in 49 individuals (32.9%) of the studied population. The allele frequencies of the C282Y and H63D were respectively 0.037 and 0.144. When we considered only the 103 individuals with relatives born in Asiago, the prevalence of the HFE mutations rose from 32.9 to 39.8%; the allele frequencies of the C282Y and H63D were respectively 0.048 and 0.174. The mean serum iron and ferritin levels were significantly higher in individuals with the HFE mutations than in normal cases. This study indicates that the prevalence of the HFE gene mutations is surprisingly high in Italians with Celtic ancestry. This could suggest the need to perform large mass studies in selected areas of the country to detect the affected patients and prevent the disease in homozygous individuals.

Successful establishment of long-term bone marrow cultures in 103 patients with myelodysplastic syndromes.

We used bone marrow biopsies instead of mononuclear cells to maintain long-term cultures from 103 patients belonging to all five sub-categories of myelodysplastic syndromes (MDS), as well as 12 normal controls. By week 4, 30-50% confluency was reached and could be maintained for up to 12 weeks with 100% confluency. The four prominent cells were fibroblasts, macrophages, endothelial cells and adipocytes. Immunohistochemical and electron microscopic studies provided lineage confirmation. Normal hematopoiesis was well supported by MDS stroma. Neither the FAB nor cytogenetics was co-related with the potency of growth. MDS stroma appears to be both morphologically and functionally normal.

Effectiveness of leukocyte interferon in patients affected by HCV-positive mixed cryoglobulinemia resistant to recombinant alpha-interferon.

OBJECTIVE: Interferon is the first-choice therapy for HCV-positive mixed cryoglobulinemia, but only a small fraction of the patients show long-term recovery from the disease. In non-responders or relapsers, the second-line therapy (high dose interferon) generally is not effective. The aim of this study was to evaluate the effectiveness of leukocyte interferon as a second-line therapy in patients who are non-responders or relapsers to a first course of recombinant interferon. METHODS: Twenty-eight patients with HCV-positive mixed cryoglobulinemia were enrolled. In each case the HCV-RNA and HCV genotype, as well as the usual laboratory parameters, were determined before, at the end of therapy and 1 year after the end of therapy. All patients were treated following the same schedule: leukocyte interferon 3,000,000 three times a week for one year. RESULTS: Only 5 patients obtained complete recovery from viral infection as well as from all signs and symptoms of the disease. Most patients (80%) experienced relief from clinical symptoms without recovery from HCV replication. Responders to the second interferon course were "relapsers" to the first treatment. No patient considered as a "non-responder" showed complete remission from the disease after the second treatment. CONCLUSIONS: A second leukocyte interferon course could be useful for patients affected by mixed crvoglobulinemia who relapsed after a first course of recombinant interferon therapy.

Measurement of mRNA expression for a variety of cytokines and its receptors in bone marrows of patients with myelodysplastic syndromes.

BACKGROUND: Myelodysplastic syndromes (MDS) are a group of disorders characterized by ineffective and dysplastic haemopoiesis. Previous studies in the lab have shown extensive apoptosis and high levels of transforming growth factor (TGF-beta) and tumor necrosis factor (TNF-alpha) in the stromal layer of MDS bone marrow. The current study focuses on the cytokines expressed in the bone marrow parenchymal cells. MATERIALS AND METHODS: Bone marrow aspirate from 5 normal donors and 26 patients with myelodysplastic syndromes were examined for mRNA expression of tumor necrosis factor alpha (TNF-alpha), macrophage colony stimulating factor (M-CSF), Flt-3 Ligand (Flt-3L), Flt-3 receptor(Flt-3 rec), interleukin 1 beta (IL 1 beta) and interleukin 1 receptor antagonist (IL-1 ra). RESULTS: Comparison of 26 MDS marrows with 5 normals showed a significantly higher value for Flt-3 rec and IL 1 beta (p = 0.031 and p = 0.031) in the former, while only Flt-1 beta rec was considerably higher (p = 0.016) in newly diagnosed patients. In previously diagnosed group, Flt-3 rec (p = 0.001), TNF-alpha (p = 0.04) and IL-1 beta (p = 0.016) were higher than normal while there was no statistically significant difference in the newly versus previously diagnosed MDS cases: CONCLUSION: mRNA expression of all six cytokines measured were considerably higher in MDS when compared to normal and that these levels tend to increase with disease duration. The precise source of these cytokines as well as their role in MDS pathogenesis remains to be determined, but this study confirms our previous reports that there is no dearth of cytokines in these bizarre myelosuppressive states.

Effects of interferon therapy on fibrosis serum markers in HCV-positive chronic liver disease.

OBJECTIVE: To evaluate serum levels of prolyl-hydroxylase and helical domain of Type IV collagen, markers of hepatic fibrogenesis, in patients with HCV-positive chronic liver disease and the effects of interferon therapy on these markers. DESIGN: Prolyl-hydroxylase and Type IV collagen were determined before therapy and each month during the treatment and follow-up. METHODS: Fifty-seven HCV-positive patients were studied. All the subjects received alpha2a recombinant interferon, 6 MU subcutaneously three times a week for 4 weeks, followed by 3 MU thrice weekly for 5 months. After cessation of treatment, each patient was followed for 12 months. Prolyl-hydroxylase and helical domain of Type IV collagen were measured by using immunoenzymatic methods. HCV-RNA and HCV genotype were determined according to the method of Okamoto. RESULTS: In the patients prolyl-hydroxylase (39.8+/-8.9 ng/ml) was not different from controls (39.1+/-5.9 ng/ml). On the contrary, the patients showed a mean Type IV collagen (133.6+/-93.3 ng/ml) significantly (P < 0.01) higher than controls (100.2+/-10.5 ng/ml). A good relationship between the degree of liver fibrosis and the Type IV collagen serum level was found (r = 0.68; P < 0.005). In both responders and non-responders the Type IV collagen levels decreased during interferon therapy. During the follow-up, in responders the Type IV collagen did not show modifications, while in non-responders/relapsers it returned rapidly to the pretreatment levels (139.1+/-100.7 ng/ml). CONCLUSION: In HCV-positive chronic liver disease, prolylhydroxylase is not a good marker of hepatic fibrosis, while Type IV collagen is a useful tool for evaluating fibrogenic activity. Interferon seems to be able to reduce the liver fibrosis even without the inhibition of viral replication and independently from liver necrosis.

Thalidomide in myelodysplastic syndromes.

The myelodysplastic syndromes are a heterogeneous group of clonal diseases of haemopoiesis, which are a challenge for both biologists and clinicians. In this paper the current classification and the recent advances in the understanding the disease mechanisms are reviewed. The recent therapeutic advances are also indicated, such as intensive and low-dose chemotherapy, new drugs, erythropoietin and colony-stimulating factors. However, the work has been focused on thalidomide, its therapeutic potential, its modes of actions, side effects, indications and future applications.

Interferon versus steroids in patients with hepatitis C virus-associated cryoglobulinaemic glomerulonephritis.

BACKGROUND/AIMS: The association between mixed cryoglobulinaemia, cryoglobulinaemic glomerulonephritis, and chronic hepatitis C virus infection has recently been described. The renal disease had usually been treated with immunosuppressive therapy, but, given the presence of viral infection, this therapy is no longer recommended. In this study, we compare steroid vs interferon therapy in a group of patients affected by hepatitis C virus-positive cryoglobulinaemic glomerulonephritis in the stationary phase. PATIENTS/METHODS: The diagnosis of cryoglobulinaemic glomerulonephritis was made bearing in mind standard criteria. Patients were randomly assigned to 2 groups receiving oral prednisone 0.2 mg/kg/die for 6 months (6 patients, group A) or lymphoblastoid interferon 3 MU, three times a week for 6 months [7 patients, group B). Hepatitis C virus-RNA was determined by reverse transcription-polymerase chain reaction and hepatitis C virus genotype according to Okamoto. Hepatitis C virus-RNA quantitation was performed by competitive polymerase chain reaction. RESULTS; The 2 groups were comparable in terms of age and severity of kidney failure. All genotypes of hepatitis C virus were found with a prevalence of Type 1b. In group A, 4 patients showed a partial response; in group B, 1 patient achieved complete remission, 4 a partial response, 2 patients in both groups showed no response. At the end of the treatment, all patients in both groups relapsed. Only 1 patient in group B became hepatitis C virus-RNA negative, and recovered from cryoglobulinaemic glomerulonephritis. CONCLUSIONS: Interferon seems to be an effective drug in the treatment of cryoglobulinaemic glomerulonephritis, but dosage and length of treatment still need to be addressed by large multicentre studies.

Human leucocyte interferon-alpha in the treatment of chronic hepatitis C.

AIM: To assess the efficacy of different schedules of human leucocyte interferon alpha in chronic hepatitis C. PATIENTS AND METHODS: A total of 213 naive patients with chronic hepatitis C were treated with 4 different schedules of human leucocyte interferon alpha. Sustained response was defined as persistently normal alanine amino transferase values with negative serum hepatitis C virus-RNA up to 12 months after therapy withdrawal. RESULTS: Rates of sustained response were 16% with 3 MU tiw for 6 months, 33% with 6 MU tiw for 5 months after a priming dose of 9 MU tiw for a month, 32% with 3 MU tiw for 12 months and 20% with 3 MU daily for 6 months. The major factors affecting the response rate were age and the hepatitis C virus genotype, as a sustained response was significantly higher in patients under 45 years and infected by hepatitis C virus types other than hepatitis C virus-1. Treatment was well tolerated and side-effects and drop-out events were similar to those described with other types of alpha-interferons. CONCLUSIONS: Human leucocyte interferon alpha appears to be equivalent to recombinant interferon-alpha in the treatment of chronic hepatitis C.

GBV-C/HGV and HCV infection in mixed cryoglobulinaemia.

Recently, a new, suspected hepatotropic virus has been identified. Named GBV-C/HGV, this virus shares with the hepatitis C virus (HCV) routes of transmission and molecular organization. Indeed, a proportion of HCV-infected patients (10-25%) are also carriers of GBV-C/HGV. Since mixed cryoglobulinaemia (MC) is closely associated with HCV infection, the aim of this study was to determine the prevalence of GBV-C/HGV infection in MC patients, and to investigate whether the double infection influenced the clinical and/or laboratory aspects of the disease. 52 patients affected by MC were studied. 100 patients affected by HCV-positive chronic liver disease (CLD) without MC were used as control group. To determine the prevalence of GBV-C/HGV infection in general population, 150 blood donors were studied, as well as 80 patients affected by non-A-E CLD. Among the MC patients, only five (9.6%) were positive for both HCV and GBV-C/HGV infection. No difference was found between patients with and without double infection as regards main clinical and laboratory aspects. Among HCV-positive CLD cases, 27 were positive for double infection. Among blood donors, the prevalence of GBV-C/HGV infection was 8.0%, whereas in cases with cryptogenetic CLD the prevalence was 5.0%. In conclusion, these data show that GBV-C/HGV infection does not play any role in the pathogenesis of MC.

Recovery from hepatitis C virus-positive cryoglobulinaemic glomerulonephritis after interferon therapy.

The association between mixed cryoglobulinaemia and chronic hepatitis C virus infection has recently been described. Cryoglobulinaemic glomerulonephritis, a complication of mixed cryoglobulinaemia, is usually treated with immunosuppressive therapy, but, given the presence of viral infection, this therapy is no longer recommended. This report concerns a case of a 30-year-old patient with cryoglobulinaemic glomerulonephritis, refractory to steroid treatment, in whom recovery from hepatitis C virus infection was obtained as well as from cryoglobulinaemic glomerulonephritis after interferon therapy. The clinical symptoms and laboratory tests were normal after prolonged interferon therapy and, 3 years after the end of treatment, the patient is free from disease.

Pilot study on the safety and efficacy of intravenous natural beta-interferon therapy in patients with chronic hepatitis C unresponsive to alpha-interferon.

BACKGROUND: Since a large fraction of patients affected by chronic hepatitis C do not respond to alpha-interferon therapy, we planned a pilot study of intravenous beta-interferon therapy in Italian patients non responsive to several courses of alpha-interferon. METHODS: Ten Italian patients with chronic hepatitis C were treated intravenously with beta-interferon to assess the biochemical and virological responses. Each patient received intravenously 6 MU of beta-interferon daily for 7 days a week for a period of 2 months; in responders, this treatment was followed by intramuscular beta-interferon administration 6 MU three times a week for an additional 8 weeks. RESULTS: All the patients were infected by the genotype 1b of hepatitis C virus and had a high serum concentration of HCV-RNA (4.1 +/- 3.3 x 10(7) copies/ml). During intravenous therapy, 4 patients (40%) showed a complete return to normal of alanino-aminotransferase and 3 cases became HCV-RNA negative. During intramuscular beta-interferon administration, two patients breakthrough. At the end of the follow-up (six months after the end of the treatment) two patients only showed return to normal of alanino-aminotransferase, but one of them remained HCV-RNA positive. CONCLUSIONS: These results indicate that even genotype 1b of hepatitis C virus can be suppressed by intravenous beta-interferon therapy, as previously described in similar cases in Japan. The rate of sustained biochemical and virologic response was, however, low, suggesting that further studies are needed to define the best regimen to achieve eradication of hepatitis C virus infection.

The clinical and biological effects of thalidomide in patients with myelodysplastic syndromes.

Thirty patients with myelodysplastic syndromes (MDS) were treated with thalidomide at 100 mg/d p.o., increased as tolerated to 400 mg/d for 12 weeks. Levels of apoptosis, macrophage number, microvessel density (MVD), tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta), interleukin 6 (IL-6), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were determined in the serum, bone marrow (BM) plasma and BM biopsies before and after therapy. Pretherapy biological characteristics of MDS patients were compared with similar studies performed in 11 normal volunteers. Ten patients demonstrated haematological improvement in the erythroid series, six becoming transfusion independent. Responders had a higher pretherapy platelet count (P < 0.048) and lower BM blasts (P < 0.013). Median time to response was 10 weeks, and four remain in remission beyond a year. Pretherapy MDS BMs showed higher MVD (P < 0.001) and TGF-beta (P < 0.03) and higher serum TNF-alpha (P < 0.008) compared with normal control subjects. After therapy, only BM TGF-beta decreased significantly (P < 0.002). Pretherapy haemoglobin was directly related to serum VEGF (P < 0.001) in responders and inversely related in non-responders (P < 0.05), suggesting the possibility that angiogenesis may be a primary pathology in the former and a consequence of anaemia-induced hypoxia in the latter. We conclude that thalidomide has important clinical and biological effects in at least a subset of MDS patients, but the precise mechanism of its action remains unknown and requires further study including a larger number of patients.

Hepatitis C virus risk: a hepatitis C virus related syndrome.

BACKGROUND: The association between mixed cryoglobulinemia (MC) and hepatitis C virus (HCV) infection has been recently described in many reports. OBJECTIVE: The aim of this study was to evaluate the long-term prognosis of hepatitis C virus-positive patients affected by mixed cryoglobulinemia with or without kidney involvement. PATIENTS: At total of 119 hepatitis C virus-positive patients affected by mixed cryoglobulinemia were divided in two groups. Group A: mixed cryoglobulinemia without kidney involvement (103 cases); group B: mixed cryoglobulinemia with glomerulonephritis (GN) (16 cases). A further 37 patients affected by mesangio-proliferative glomerulonephritis (MPGN) were evaluated as controls (group C). METHODS: Anti-hepatitis C virus antibodies were determined by commercial kits and hepatitis C virus-RNA was detected by polymerase chain reaction (PCR) amplification of the 5' untranslated region (5'UTR) of the virus. The hepatitis C virus genotype was determined according to Okamoto. Liver biopsy was performed in 62 patients, bone marrow biopsy in 65 patients, and kidney biopsy in all patients with proteinuria. RESULTS: In group A, 46 patients (45%) were affected by chronic liver disease (CLD), 21 (20%) by low-grade non-Hodgkin's lymphoma (NHL) and 16 (15%) by both diseases. All patients of group B were affected by type I membrano-proliferative glomerulonephritis, 3 (19%) by chronic liver disease, 6 (37%) by low-grade non-Hodgkin's lymphoma, and 7 (44%) by both diseases. Several genotypes of hepatitis C virus were found, but Type 1b was prevalent. In group C, no patient showed chronic liver disease or non-Hodgkin's lymphoma. Younger age, higher mean blood pressure, lower C4 serum level, and poorer survival significantly distinguished group B from group A. Survival rates at 5 years were: 87.4% for group A, 89.5% for group C, and 50.0% for group B. None of the patients of group B developed kidney failure requiring dialysis, whilst infections were the leading cause of death. CONCLUSIONS: In hepatitis C virus-positive patients, the presence of mixed cryoglobulinemia associated with kidney involvement seems to indicate a new syndrome characterized by immune system impairment, lack of progression to kidney failure, and poor survival (hepatitis C virus-Risk syndrome).

Intramedullary apoptosis of hematopoietic cells in myelodysplastic syndrome patients can be massive: apoptotic cells recovered from high-density fraction of bone marrow aspirates.

A higher percentage of apoptotic cells (apoptotic index or AI) is consistently found in bone marrow (BM) biopsies compared to BM aspirates of patients with myelodysplastic syndrome (MDS). Most studies have only investigated the low-density fraction (LDF) mononuclear cells from BM aspirates following density separation for AI determination. In the present study, both LDF and high-density fraction (HDF) cells for AI were examined by electron microscopy (EM) in 10 MDS patients and 4 healthy donors. Matched BM biopsies were subjected to AI detection by in situ end labeling (ISEL) of fragmented DNA. The results indicate that in LDF and HDF cells, AI is consistently higher in MDS patients (8.5% vs 1.5%, respectively; P =.039) compared to healthy donors (27% vs 4%, respectively; P =. 004). The BM biopsy AI was also higher in MDS patients than in healthy donors (3+ vs 0+, respectively; P =.036). In addition, in MDS patients, more apoptotic cells were found in HDF cells than in LDF cells (27% vs 8.5%, respectively;P =.0001). All stages of maturation, ranging from blasts to terminally mature cells belonging to all 3 lineages, were represented in the dying cells in both compartments. Using EM, typical Pelger-Huett-type cells appeared to be apoptotic granulocytes. Both LDF and HDF cells should be examined for an accurate estimation of apoptotic cells because AI would be underestimated if only the LDF cells were studied. Ultrastructural studies consistently show a higher AI in BM biopsies compared to BM aspirates despite the correction factor of HDF cells provided by AI. This may represent the actual extant state, which could conceivably be due to a higher concentration of proapoptotic signals in the biopsies. (Blood. 2000;96:1388-1392)

Ethnic difference in the prevalence of monoclonal B-cell proliferation in patients affected by hepatitis C virus chronic liver disease.

BACKGROUND/AIM: In previous studies we demonstrated that all patients affected by HCV-positive type II mixed cryoglobulinaemia have a monoclonal B-cell population in peripheral blood mononuclear cells, and that a large fraction of HCV-infected patients develop a monoclonal B-cell expansion, even in the absence of dosable serum cryoglobulins. However, the prevalence of Type II mixed cryoglobulinaemia in HCV-infected individuals seems to be high in Italy, whereas it is very low in Japan. This study was performed to investigate whether there are ethnic differences in the prevalence of asymptomatic HCV-associated monoclonal B-cell expansions. METHODS: Forty-four Japanese patients affected by HCV-positive chronic liver disease (two healthy carriers, 31 chronic hepatitis and 11 cirrhosis) were compared with a group of 60 Italian patients (one healthy carrier, 49 chronic hepatitis, and 10 cirrhosis) without dosable levels of cryoglobulins. The monoclonality of peripheral blood mononuclear cells was investigated by RT/PCR analysis of Immunoglobulin gene rearrangements. Liver function tests, rheumatoid factor, cryocrit level, anti-HCV antibodies, HCV-RNA, and HCV genotype were performed according to standard methodology. RESULTS: A B-cell monoclonal population was found in 26% of Italian patients, whereas all Japanese patients were negative. No correlation was found between B-cell monoclonality and severity of liver disease, length or source of the infection, HCV genotype, sex, clinical and biochemical parameters. CONCLUSIONS: This study indicates that a monoclonal B-cell proliferation in peripheral blood mononuclear cells is common in HCV infection, but only in Italy, whereas it is absent in Japan. This explains the very low prevalence of Type II mixed cryoglobulinaemia in HCV-positive Japanese subjects, and suggests that HCV is able to determine a B-cell expansion only in the presence of, presently undetermined, host factors.

Thalidomide produces transfusion independence in long-standing refractory anemias of patients with myelodysplastic syndromes.

Thalidomide was administered to 83 patients with myelodysplastic syndrome (MDS), starting at 100 mg by mouth daily and increasing to 400 mg as tolerated. Thirty-two patients stopped therapy before 12 weeks (minimum period for response evaluation), and 51 completed 12 weeks of therapy. International Working Group response criteria for MDS were used to evaluate responses. Intent-to-treat (ITT) analysis classified all off-study patients as nonresponders. Off-study patients belonged to a higher risk category (P =.002) and had a higher percentage of blasts in their pretherapy bone marrow than patients who completed 12 weeks of therapy (P =.003). No cytogenetic or complete responses were seen, but 16 patients showed hematologic improvement, with 10 previously transfusion-dependent patients becoming transfusion independent. Responders had lower pretherapy blasts (P =.016), a lower duration of pretherapy platelet transfusions (P =.013), and higher pretherapy platelets (P =.003). Among responders, 9 had refractory anemia (RA); 5 had RA with ringed sideroblasts; and 2 had RA with excess blasts. By ITT analysis, 19% of patients (16 of 83) responded, and when only evaluable patients were analyzed, 31% (16 of 51) responded. It was concluded that thalidomide, as a single agent, is effective in improving cytopenias of some MDS patients, especially those who present without excess blasts. (Blood. 2001;98:958-965)

The clinical and biologic significance of abnormal lipid profiles in patients with myelodysplastic syndromes.

Serum lipid profiles were obtained in 108 patients with myelodysplastic syndrome (MDS) and compared to 28 healthy volunteers. Serum cholesterol and low-density and high-density lipoproteins (LDL and HDL) were found to be significantly lower in MDS patients than in normals (p = 0.0001, 0.0038 and 0.037, respectively). This difference was significant for all MDS categories. Serum cholesterol and HDL were negatively related to biopsy cellularity (p = 0.001 and 0.0001, respectively), and serum triglycerides were negatively related to labeling index (p = 0.0003). No differences were noted in the lipid profiles of MDS patients with normal versus abnormal karyotypes. However, low-risk MDS patients with abnormal karyotypes had significantly lower triglyceride levels compared with the high-risk patients (p = 0.027), as did low-risk patients with normal cytogenetics (p = 0.015). Serum HDL levels were significantly higher for the low-risk group with normal cytogenetics as well (p = 0.003). We conclude that serum cholesterol, LDL, and HDL are significantly reduced in MDS patients, probably indicating excessive intracellular lipid biosynthesis in the expanding clone. These relatively simple measurements could serve as important prognostic markers and reliable indicators of disease activity in individual patients. Prospective studies to determine their utility as independent variables that guide the need for active therapeutic intervention are warranted.

Peripheral blood neutrophils from hepatitis C virus-infected patients are replication sites of the virus.

BACKGROUND AND OBJECTIVE: Hepatitis C virus (HCV) is able to cause not only acute and chronic liver disease, but also immunologic and hematologic disorders. In order to clarify the extra-hepatic tropism of HCV, and to understand the pathogenetic mechanisms of HCV infection, we evaluated viral replication in peripheral blood mononuclear cells. DESIGN AND METHODS: The presence of genomic and antigenomic (replicative) forms of HCV in B- and T-lymphocytes, monocytes, and polymorphonuclear leukocytes (PML) was determined by reverse transcriptase-polymerase chain reaction in 54 HCV-RNA positive patients and, as control groups, in 10 patients who had recovered from HCV infection without evidence of serum HCV-RNA, and in 10 HCV-negative subjects. RESULTS: In HCV-RNA positive patients, the genomic RNA was found in 94% of B-cells, in 14% of T-cells, in 40% of monocytes and in 77% of PML, while only 1 of the HCV-RNA negative subjects showed positivity in B-cells. The anti-genomic form of HCV-RNA was found in 52% of B-cells, in 3% of monocytes, and in 31% of PML. By contrast, it was never detected in T-cells and in HCV-RNA negative subjects. Neither genomic nor anti-genomic forms were found in HCV-negative cases. INTERPRETATION AND CONCLUSIONS: These data suggest that PML are replication sites of HCV. Whether the infection occurs at the level of the stem cells or subsequently during myeloid cell differentiation is, as yet, unknown. The absence of correlation between the presence of replicative forms and any clinical and/or laboratory data opens the question of the role of HCV replication in extra-hepatic sites.

Cryoglobulinaemic membranoproliferative glomerulonephritis and hepatitis C virus infection.

BACKGROUND/AIM: A striking correlation between mixed cryoglobulinaemia and chronic hepatitis C virus infection has recently been described. Since membrano-proliferative glomerulonephritis is a rare complication of mixed cryoglobulinaemia, this study was undertaken to determine the prevalence of Hepatitis C virus infection in membrano-proliferative glomerulonephritis. PATIENTS: Eighteen patients, selected among a group of 121 affected by mixed cryoglobulinaemia, with renal involvement were included in the present study. A group of 148 patients affected by renal disease of different aetiology and the general population (6,917 people) were used as control groups. METHODS: The presence of anti-hepatitis C virus antibodies was determined by a commercial kit. The hepatitis C virus genotype was determined according to Okamoto. All patients underwent kidney and bone marrow biopsy, while the hepatic biopsy was performed in those showing signs of chronic liver disease. RESULTS: In patients with renal involvement, the kidney biopsy showed the presence of membrano-proliferative glomerulonephritis Type I in all cases. Chronic liver disease was present in eleven patients (61%). All patients were positive for serum hepatitis C virus-RNA. Bone marrow biopsy was normal in five cases, while in the others paratrabecular foci of infiltration by small lymphocytes were present. In six of these, the massive bone marrow infiltration by lymphoplas-macytoid lymphocytes suggested the diagnosis of low grade non-Hodgkin's lymphoma. In the group of patients affected by other chronic renal disease, the prevalence of hepatitis C virus infection (3.1%) was not different from that of the general population (3.2%). CONCLUSIONS: Hepatitis C virus seems to be the aetiologic agent of mixed cryoglobulinaemia and, consequently, of membrano-proliferative glomerulonephritis.