MBL2 Genetic Variants in HCV Infection Susceptibility, Spontaneous Viral Clearance and Pegylated Interferon Plus Ribavirin Treatment Response.

Hepatitis C is disease that damages the liver, and it is caused by the hepatitis C virus (HCV). The pathology became chronic in about 80% of the cases due to virus persistence in the host organism. The standard of care consists of pegylated interferon plus ribavirin; however, the treatment response is very variable and different host/viral factors may concur in the disease outcome. The mannose-binding protein C (MBL) is a component of the innate immune system, able to recognize HCV and consecutively activating the immune response. MBL is encoded by MBL2 gene, and polymorphisms, two in the promoter region (H/L and X/Y) and three in exon 1 (at codon 52, 54 and 57), have been described as functionally influencing protein expression. In this work, 203 Italian HCV patients and 61 healthy controls were enrolled and genotyped for the five MBL2 polymorphisms mentioned above to investigate their role in HCV infection susceptibility, spontaneous viral clearance and treatment response. MBL2 polymorphisms were not associated with HCV infection susceptibility and with spontaneous viral clearance, while MBL2 O allele, O/O genotype, HYO haplotype and DP combined genotype (all correlated with low or deficient MBL expression) were associated with sustained virological response. Moreover, a meta-analysis to assess the role of MBL2 polymorphisms in HCV infection susceptibility was also performed: YA haplotype could be associated with protection towards HCV infection.

Engineered mucoperiosteal scaffold for cleft palate regeneration towards the non-immunogenic transplantation.

Cleft lip and palate (CL/P) is the most prevalent craniofacial birth defect in humans. None of the surgical procedures currently used for CL/P repair lead to definitive correction of hard palate bone interruption. Advances in tissue engineering and regenerative medicine aim to develop new strategies to restore palatal bone interruption by using tissue or organ-decellularized bioscaffolds seeded with host cells. Aim of this study was to set up a new natural scaffold deriving from a decellularized porcine mucoperiosteum, engineered by an innovative micro-perforation procedure based on Quantum Molecular Resonance (QMR) and then subjected to in vitro recellularization with human bone marrow-derived mesenchymal stem cells (hBM-MSCs). Our results demonstrated the efficiency of decellularization treatment gaining a natural, non-immunogenic scaffold with preserved collagen microenvironment that displays a favorable support to hMSC engraftment, spreading and differentiation. Ultrastructural analysis showed that the micro-perforation procedure preserved the collagen mesh, increasing the osteoinductive potential for mesenchymal precursor cells. In conclusion, we developed a novel tissue engineering protocol to obtain a non-immunogenic mucoperiosteal scaffold suitable for allogenic transplantation and CL/P repair. The innovative micro-perforation procedure improving hMSC osteogenic differentiation potentially impacts for enhanced palatal bone regeneration leading to future clinical applications in humans.

Fibronectin gene polymorphisms are associated with the development of B-cell lymphoma in type II mixed cryoglobulinemia.

OBJECTIVE: To analyse fibronectin (FN) gene polymorphisms in type II mixed cryoglobulinemic syndrome (MCsn), an immune-complex mediated systemic vasculitis linked to hepatitis C virus (HCV) infection and characterised by rheumatoid factor (RF) positive B-cell proliferation at high risk for the progression into non-Hodgkin's lymphoma (NHL). METHODS: Samples from 74 patients with MCsn (type II serum cryoglobulins and clinical signs of vasculitis) were studied. In all, 58 (78.4%) patients were HCV-positive. In total, 21 (28.4%) patients developed a B-cell NHL during the course of MCsn. A total of 72 patients with HCV-negative and MC-unrelated NHL and 110 healthy blood donors (HBDs) were taken as controls. HaeIIIb and MspI FN gene polymorphisms were analysed by PCR and specific restriction enzyme digestions, following reported procedures. Plasma FN levels were analysed by ELISA, whenever possible. RESULTS: HaeIIIb and MspI allele and genotype frequencies did not differ between MCsn patients and HBDs. Of note, the DD-MspI (OR = 5.99; CI 1.77-20.261, p = 0.0039) and the AA-HaeIIIb (OR = 4.82, CI 1.42-16.39, p = 0.0176) homozygosis appeared significantly associated with the development of B-cell NHL in MCsn patients, with the HaeIIIb A allele possibly conferring an increased risk of NHL in the general population (OR = 1.72, CI 1.128-2.635, p = 0.0133). None of the other MCsn-related clinical manifestations were significantly associated with a particular genetic pattern. No association between FN plasma levels and FN genotypes was found. CONCLUSION: Genotyping for MspI and HaeIIIb FN gene polymorphisms may be clinically relevant to define the risk of lymphoma development in MCsn.

[Fibronectin gene polymorphisms and clinical manifestations of mixed cryoglobulinemic syndrome: increased risk of lymphoma associated to MspI DD and HaeIII AA genotypes]. / Polimorfismi del gene della fibronectina e manifestazioni cliniche della sindrome crioglobulinemica: aumentato rischio di linfoma legato ai genotipi MspI DD e HaeIII AA.

OBJECTIVE: To analyse FN gene polymorphisms in type II mixed cryoglobulinemic syndrome (MCsn), an immune-complex mediated systemic vasculitis linked to hepatitis C virus (HCV) infection and characterized by rheumatoid factor (RF) positive B-cell proliferation at high risk for the progression into non Hogkin's lymphoma (NHL). METHODS: Samples from eighty-one patients, with MCsn (type II serum cryoglobulins and clinical signs of vasculitis were studied. Sixty-five (65/81, 80.3%) patients were HCV-positive. Twenty-one (25.9%) patients had developed a B-cell NHL during the course of MCsn. Seventy-two patients with HCV-negative and MC-unrelated NHL and 110 healthy blood donors (HBDs) were taken as controls. HaeIIIb and MspI FN gene polymorphisms were analysed by ELISA, whenever possible. RESULTS: HaeIIIb and MspI allele and genotypic frequencies did not differ between MCsn patients and HBDs. Of note, the DD-MspI allele and genotype frequencies did not differ between MCsn patients and HBDs. Of note, the DD-MspI (OR = 5.56; DI = 1.67-18.51, p = 0.0046) and the AA-HaeIIIb (OR = 5.54, CI = 1.64- 18.76, p = 0.0066) homozygosis appeared significantly and independently associated with the development of B-cell NHL in MCsn patients, with the HaeIIIbA allele possibly conferring an increased risk of NHL in the general population (OR = 1.72, CI = 1.128-2.635, p = 0.0133). In contrast, the major vasculitic manifestations, such as peripheral neuropathy, skin ulcers and glomerulonephritis tended to be associated with the counterpart MspI C allele. No association between FN plasma levels and FN genotypes was found. CONCLUSION: Genotyping for MspI and HaeIIIb FN gene polymorphisms may be clinically relevant to define the predisposition to the major clinical manifestations in MCsn.

NOTCH1 mutations are associated with high CD49d expression in chronic lymphocytic leukemia: link between the NOTCH1 and the NF-κB pathways.

In chronic lymphocytic leukemia (CLL), stabilizing mutations of NOTCH1, affecting up to 10-15% of cases, have been associated to poor prognosis, disease progression and refractoriness to chemotherapy. NOTCH1 mutations are significantly overrepresented in trisomy 12 CLL, a disease subset frequently expressing CD49d, the α4 chain of the very-late-activation-4 integrin, a well-known key regulator of microenviromental interactions, and negative prognosticator in CLL. In the present study, by analysing a wide cohort of 1180 CLL, we observed a very strong association between the presence of NOTCH1 mutations and the expression of CD49d (P<0.0001), occurring also outside the trisomy 12 CLL subset. Using both the MEC-1 CLL-like cells stably transfected with the NOTCH1 intracellular domain and primary CLL cells bearing a mutated or wild-type NOTCH1 gene configuration, we provide evidence that triggering of the NOTCH1 pathway resulted in a positive CD49d expression regulation, which was driven by a NOTCH1-dependent activation of nuclear factot-κB (NF-κB). Consistently, pharmacological inhibition of the NOTCH1 and/or of the NF-κB pathways resulted in impaired NF-κB nuclear translocation with consequent down-modulation of CD49d expression. Altogether, our data link for the first time NOTCH1 mutations to CD49d expression regulation through the involvement of the NF-κB pathway in CLL.

IgM-producing chronic lymphocytic leukemia cells undergo immunoglobulin isotype-switching without acquiring somatic mutations.

The malignant B cells in chronic lymphocytic leukemia (CLL) typically express low-density membrane IgM or IgM/IgD. In vitro experiments have shown that the CLL cells can be induced to differentiate into cells that secrete immunoglobulin (Ig) and can occasionally undergo heavy (H) chain class switching. We now show that the CLL cells also undergo isotype-switching in vivo, since gamma and/or alpha H chain transcripts with identical FW3/CDR3/FW4 regions as the mu CLL transcripts were detected in all of the 13 investigated patients with IgM+ CLL. In most cases switching had occurred to alpha1 and gamma3, but CLL transcripts corresponding to the other gamma chain isotypes were also detected. In one case both the productively and nonproductively rearranged allele were found to undergo H chain class switching. CLL gamma transcripts were also present in surface IgG+ sorted B cells, demonstrating that a small subset of the CLL cells express membrane IgG. In addition, transcripts encoding secretary gamma2 and gamma3 H chains were detected in two cases, which suggests that some serum IgG could be produced by the leukemic clone. Analysis of sorted PBL showed that isotype-switching occurs in CLL cells that express the CD5 antigen. Finally, nucleotide sequence analysis showed that the mu, alpha, and gamma CLL transcripts are identical, demonstrating that the CLL cells do not accumulate somatic mutations in their variable region genes after the H chain class switching. These data provide in vivo evidence that isotype-switching is a frequent phenomenon in CLL, and indicate that a subset of the CLL lymphocytes progress to later stages of B cell differentiation.

Favorable tissue effects of quantum molecular resonance device (Vesalius) compared with standard electrocautery. A novel paradigm in lung surgery.

BACKGROUND: Electrosurgical devices are largely employed in thoracic surgery but their use is burdened by extensive necrosis and second intention healing. METHODS: A rat model of thoracotomy was performed on 46 adult male rats using a standard electrocautery or a new quantum molecular resonance (QMR) instrument called Vesalius. Skin, muscle and lung specimens were obtained immediately and 2 weeks after surgery to evaluate acute and late effects. RESULTS: Both in the short- and long-term study, Vesalius produced less severe tissue damage than that of standard electrocautery. CONCLUSIONS: The use of the QMR device may provide an alternative to gold-standard electrosurgical devices in thoracic surgery.

HLA-G regulatory polymorphisms are associated with susceptibility to HCV infection.

BACKGROUND: Hepatitis C virus (HCV) is able to bypass the immune system modulating innate and adaptive immune response and blocking T helper 1 (Th1) cell production. Because the human leukocyte antigen (HLA)-G molecule has immunomodulatory properties inhibiting the function and production of natural killer and cytotoxic lymphocyte T cells, as well as promoting shift from Th1 toward Th2 response, we hypothesized its involvement in susceptibility to HCV infection. MATERIALS AND METHODS: Considering that HLA-G mRNA expression has been reported to be under genetic control, an association study was conducted analyzing 800 base pairs upstream the ATG at the 5'upstream regulator region (URR) and 850 base pairs from ATG to exon 3 and the 3'untranslated region (UTR) of HLA-G gene in Italian HCV-positive patients and uninfected controls. RESULTS: Four 5'URR polymorphisms (-725C>G>T, -509C>G, -400G>A and -398G>A), 7 polymorphisms at coding region (+15G>A, +36G>A, +243G>A, insC506, 531G>C, delA615 and 685G>A), the +644G>T polymorphism, and 1 haplotype (TTGTTCCIGAC) showed different frequency distributions between HCV patients and uninfected controls. CONCLUSION: The results from our study suggest a possible involvement of HLA-G in the risk modulation toward HCV infection.

NOTCH1-mutated chronic lymphocytic leukemia cells are characterized by a MYC-related overexpression of nucleophosmin 1 and ribosome-associated components.

In chronic lymphocytic leukemia (CLL), the mechanisms controlling cell growth and proliferation in the presence of NOTCH1 mutations remain largely unexplored. By performing a gene expression profile of NOTCH1-mutated (NOTCH1-mut) versus NOTCH1 wild-type CLL, we identified a gene signature of NOTCH1-mut CLL characterized by the upregulation of genes related to ribosome biogenesis, such as nucleophosmin 1 (NPM1) and ribosomal proteins (RNPs). Activation of NOTCH1 signaling by ethylenediaminetetraacetic acid or by coculture with JAGGED1-expressing stromal cells increased NPM1 expression, and inhibition of NOTCH1 signaling by either NOTCH1-specific small interfering RNA (siRNA) or γ-secretase inhibitor reduced NPM1 expression. Bioinformatic analyses and in vitro activation/inhibition of NOTCH1 signaling suggested a role of MYC as a mediator of NOTCH1 effects over NPM1 and RNP expression in NOTCH1-mut CLL. Chromatin immunoprecipitation experiments performed on NOTCH1 intracellular domain (NICD)-transfected CLL-like cells showed the direct binding of NOTCH1 to the MYC promoter, and transfection with MYC-specific siRNA reduced NPM1 expression. In turn, NPM1 determined a proliferation advantage of CLL-like cells, as demonstrated by NPM1-specific siRNA transfection. In conclusion, NOTCH1 mutations in CLL are associated with the overexpression of MYC and MYC-related genes involved in protein biosynthesis including NPM1, which are allegedly responsible for cell growth and/or proliferation advantages of NOTCH1-mut CLL.

NOTCH1 mutations associate with low CD20 level in chronic lymphocytic leukemia: evidence for a NOTCH1 mutation-driven epigenetic dysregulation.

In chronic lymphocytic leukemia (CLL), NOTCH1 mutations have been associated with clinical resistance to the anti-CD20 rituximab, although the mechanisms behind this peculiar behavior remain to be clarified. In a wide CLL series (n=692), we demonstrated that CLL cells from NOTCH1-mutated cases (87/692) were characterized by lower CD20 expression and lower relative lysis induced by anti-CD20 exposure in vitro. Consistently, CD20 expression by CLL cells was upregulated in vitro by γ-secretase inhibitors or NOTCH1-specific small interfering RNA and the stable transfection of a mutated (c.7541-7542delCT) NOTCH1 intracellular domain (NICD-mut) into CLL-like cells resulted in a strong downregulation of both CD20 protein and transcript. By using these NICD-mut transfectants, we investigated protein interactions of RBPJ, a transcription factor acting either as activator or repressor of NOTCH1 pathway when respectively bound to NICD or histone deacetylases (HDACs). Compared with controls, NICD-mut transfectants had RBPJ preferentially complexed to NICD and showed higher levels of HDACs interacting with the promoter of the CD20 gene. Finally, treatment with the HDAC inhibitor valproic acid upregulated CD20 in both NICD-mut transfectants and primary CLL cells. In conclusion, NOTCH1 mutations are associated with low CD20 levels in CLL and are responsible for a dysregulation of HDAC-mediated epigenetic repression of CD20 expression.

CD49d prevails over the novel recurrent mutations as independent prognosticator of overall survival in chronic lymphocytic leukemia.

CD49d, the alpha-chain of the integrin heterodimer α4ß1, was identified among the strongest predictors of overall survival (OS) in chronic lymphocytic leukemia (CLL), along with IGHV mutational status and deletion of the 17p chromosome involving TP53. In addition to TP53, the clinical relevance of NOTCH1, SF3B1 and BIRC3 gene mutations has been recently emphasized. By analyzing a cohort of 778 unselected CLL patients, we assessed the clinical relevance of CD49d as an OS predictor in subgroups defined by mutation/deletion of the TP53, NOTCH1, SF3B1 and BIRC3 genes. In this context, CD49d emerged as an independent predictor of OS in multivariate Cox analysis (Hazard ratio =1.88, P<0.0001). Consistently, high CD49d expression identified CLL subsets with inferior OS in the context of each category of a previously reported hierarchical risk stratification model. Moreover, by evaluating the relative importance of biological prognosticators by random survival forests, CD49d was selected among the top-ranked OS predictor (variable importance =0.0410), along with IGHV mutational status and TP53 abnormalities. These results confirmed CD49d as an independent negative OS prognosticator in CLL also in comprehensive models comprising the novel recurrent mutations. In this context, TP53 disruption and NOTCH1 mutations retained prognostic relevance, in keeping with their roles in CLL cell immuno-chemoresistance.

Hepatitis C virus and non-Hodgkin's lymphoma 10 years later.

Hepatitis C virus is associated with chronic liver disease as well as with lymphoproliferative disorders such as mixed cryoglobulinemia and, likely, non-Hodgkin's lymphomas. The association between hepatitis C virus infection and B-cell lymphoma is controversial since it shows a strong regional variation. In fact, the prevalence of hepatitis C virus infection in non-Hodgkin's lymphoma shows a prevalence ranging between 7.4 and 37.0%. However, the intimate pathogenetic mechanism involved in hepatitis C virus-associated lymphomas remains considerably unknown. Hepatitis C virus may exert its oncogenic potential via an indirect mechanism or utilise other pathways directly. It is reasonable to assume that several different pathogenetic mechanisms operate in the wide spectrum of hepatitis C virus-related lymphoproliferative disorders, which include the intermediate to high-grade lymphoma, and the more common indolent, low-grade lymphoma, preceded by long standing symptomatic mixed cryoglobulinemia Type II. In this review, the etiopathogenetic role of hepatitis C virus in non-Hodgkin's lymphoma is discussed on the basis of molecular, clinical and epidemiological considerations. The management of hepatitis C virus-associated non-Hodgkin's lymphoma is similar to that of conventional lymphoma, although viral reactivation or the underlying chronic liver disease can complicate chemotherapy. Whether to treat low-grade hepatitis C virus-related lymphomas with anti-viral therapy is still debatable, but encouraging data emerge from some recent studies.

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.

Ethanol metabolism and aging: the role of "first pass metabolism" and gastric alcohol dehydrogenase activity.

To investigate the effect of age and gender on ethanol metabolism, first-pass metabolism (FPM) and gastric alcohol-dehydrogenase (ADH) activity were compared in 32 elderly and 30 young adult nonalcoholic subjects. The FPM was obtained from the difference between the area under the curve of ethanol blood concentration after intravenous or oral administration of ethanol 0.3 g/Kg b.w. The ADH activity was determined in samples of gastric mucosa obtained during diagnostic endoscopy. In the young adult group the FPM was higher in men than in women (3.3 +/- 2.3 vs 1.2 +/- 0.9 mmol/l/h, respectively, p < .01). In aged subjects FPM was found to be very low for men (1.1 +/- 0.8 mmol/l/h, p < .001); conversely, FPM was not significantly reduced in women (1.7 +/- 0.8 mmol/l/h, p = n.s.). The gastric ADH activity was significantly (p < .01) higher in young adult men than women, whereas in aged subjects the activities were low (p < .0001) in both sexes. Thus, gender-related FPM differences equalize in the elderly or are even reversed, most likely because of gastric mucosal atrophy, which occurs more in men than women.

Hepatitis C virus and non-Hodgkin's lymphomas.

The etiology of non-Hodgkin's lymphomas (NHL) remains a controversial matter, but, in the last few years, considerable evidence suggests that aberrations of the immune system and viruses may act as etiologic agents, in at least some cases of NHL. In fact, patients with primary immuno-deficiencies, or those suffering from diseases characterized by autoimmune dysfunction, show an increased risk for the development of NHL. Several viruses have been identified as possible etiologic agents for NHL; one of the best studied is the Epstein-Barr virus, which was detected in cultures of tumor cells from patients with Burkitt's lymphoma. The pathogenetic potential of this virus is illustrated by its association with an increasing number of malignant diseases. In addition, the human T-cell lymphotropic virus family (HTLV), was also recognized as possible etiologic agents for several lymphomas, such as cutaneous T-cell lymphoma and T-cell leukemia-lymphoma syndrome (HTLV-I), and T-cell hairy cell leukemia (HTLV-II). Recently, the presence of hepatitis C virus infection has also been recognized in several hematological malignancies such as mixed cryoglobulinemia, low-grade malignant lymphomas and Waldenström's disease. The possible etiopathogenetic role of this virus in non-Hodgkin's lymphomas is discussed on the basis of molecular, clinical, and epidemiological considerations.

Hepatic glutathione determination after ethanol administration in rat: evidence of the first-pass metabolism of ethanol.

As a fraction of ingested ethanol is metabolized by gastric mucosa, different amounts of alcohol should reach the liver when the same dose is administered by oral or intravenous route. Therefore, we investigated the time-course of hepatic reduced glutathione (GSH) concentrations after intra-peritoneal or intra-gastric load of the same amount of ethanol in the rat. The test was also performed in fasted and Cimetidine-treated rats. The oral ethanol administration was followed by a less pronounced decrease and by a quicker recovery of hepatic content of GSH than after intraperitoneal route. In the fasted rat, basal hepatic GSH significantly decreased; after alcohol administration the decrease of hepatic GSH was more severe and prolonged than in the fed rat. Cimetidine was shown to be a potent inhibitor of gastric ADH. Pre-treatment with Cimetidine did not change the basal levels of hepatic GSH, but after oral ethanol load, the decrease of the hepatic GSH content was significantly (p < 0.005) more pronounced than in controls. This study demonstrates the beneficial effects of gastric ethanol metabolism on the liver. The reduced gastric ethanol metabolism, induced by fasting or by Cimetidine resulted in a decreased content and delayed recovery of liver GSH content.

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.

Effects of two different alpha-interferon regimens on clinical and virological findings in mixed cryoglobulinemia.

OBJECTIVE: As previous studies have shown a good response of mixed cryoglobulinemia (MC) to alpha-interferon (IFN) therapy, we investigated the efficacy and tolerability of two IFN regimens in a group of 36 patients affected by MC. METHODS: The patients, diagnosed on the basis of standard clinical and laboratory criteria, were randomly divided into 2 groups: group A (18 cases) received alpha 2b-IFN 3 M.U. thrice a week for six months, while group B (18 cases) received alpha 2b-IFN thrice a week for 1 year. The patients were followed for six months after the end of therapy. Liver function tests, cryoglobulin determinations and a clinical examination were performed each month. HCV serology and HCV-RNA detection by PCR were performed before therapy and at the end of the follow-up period. RESULTS: The two study groups were comparable in age, male/female ratio, purpura score, cryoglobulin level, mean ALT serum activity and liver histology. 32 patients (89%) were positive for anti-HCV antibodies and 29 (81%) for HCV-RNA. During therapy all patients showed a significant (p < 0.001) decrease in their cryoglobulin level as well as improvement (p < 0.05) in their purpura score. In group A, five patients (28%) showed normalized ALT, but three later relapsed. In group B seven patients (39%) responded to treatment but three relapsed after suspension of the drug. Two patients from group B developed severe side effects (hypothyroidism and depression) and therapy was discontinued after 9 and 11 months, respectively. In all the non-responders and relapsed patients, purpura, ALT, and cryoglobulins rose to pre-treatment levels within a few months. At the end of follow-up, two patients from group A (11%) and four in group B (22%) had achieved complete remission. CONCLUSION: This study indicates that IFN is useful in MC and that viral replication can be considered the target of the therapy. Despite the absence of a statistical difference in the response rate between the two regimens (due to the low number of subjects), the one-year therapy course seemed to show a better efficacy, although associated with higher toxicity.

HCV and lymphoproliferative diseases.

METHODS: Genomic and replicative forms of HCV-RNA in B lymphocytes were detected by RT-PCR, and HCV genotyping was performed using universal and type-specific primers for the core region. Immunoglobulin gene rearrangement was detected by RT-PCR. RESULTS: The presence of genomic and replicative forms of HCV-RNA in the 5'NC region was investigated on total RNA extracted from subpopulations of PBMC. The frequency of HCV-RNA was higher in the B lymphocytes than in other PBMC. In two patients a larger sized band was present in the B lymphocytes and PMN; this band could represent either another form of HCV-RNA or a cross-reaction between cellular RNA and HCV primers. HCV-RNA detected using primers for the core region was negative in the patients examined. Immunoglobulin monoclonal gene rearrangement was present on the cDNA in all of the HCV and type II cryoglobulinemia positive samples except two; in contrast, it was absent in the HCV positive and cryoglobulinemia negative samples. The analysis of immunoglobulin monoclonal gene rearrangement on DNA showed the presence of new positive samples among the HCV positive, type II cryoglobulinemia negative patients, who had been negative when PCR was performed on cDNA. Denaturing sequencing gel showed clearer results than agarose gel. CONCLUSIONS: The early detection of immunoglobulin monoclonal gene rearrangement and expression is very important because it could provide evidence of the possible lymphoproliferative evolution of HCV infection. In addition, these investigations together with PCR product sequencing could show us the steps in the clonal selection of B lymphocytes towards malignant transformation, in which HCV plays a direct and/or indirect role.

HCV genotypes and cryoglobulinemia.

OBJECTIVE: To evaluate the HCV genotype distribution in subjects affected by cryoglobulinemia in order to verify its possible role in the pathogenesis of the disease and to provide the clinician with a useful datum for therapy. METHODS: Nested PCR with universal and type-specific primers was used for the genotyping. RESULTS: Genotype I (1a) was never present in cryoglobulinemia, while it was present in 7 (4.3%) patients with chronic hepatopathy and in 4 (10.8%) asymptomatic patients. Type II (1b) was present in 28 (58.3%) and in 8 (47.1%) cryoglobulinemic patients with and without hepatopathy, respectively, in 106 (64.6%) patients with chronic hepatitis; in one patient with acute hepatitis; and in 14 (37.9%) asymptomatic patients. Type III (2a) was present in 2 (4.2%) and 2 (11.8%) cryoglobulinemic patients with and without hepatopathy, respectively; in 1 (0.6%) patient with chronic hepatopathy; and in 2 (5.4%) asymptomatic subjects. Type IV (2b) was present in 1 (2.1%) and in 2 (11.8%) cryoglobulinemic patients with and without hepatopathy, respectively; in 5 (3%) patients with chronic hepatopathy; and in 1 (2.7%) asymptomatic subject. Coinfections were present in 42 cases: 6 (12.5%) cryoglobulinemia with hepatopathy, 4 (23.5%) cryoglobulinemia without hepatopathy, 25 (15.3%) chronic hepatopathy, and in 7 (18.9%) asymptomatic subjects. For 41 (15.4%) strains typing was not possible. Eight of the "untypable" strains and 3 strains from patients with coinfection proved to belong to a new genotype. CONCLUSIONS: Genotype II (1b) was the most frequent in patients with and without cryoglobulinemia; genotype I (1a) was absent in all 65 patients with cryoglobulinemia, in whom, however, as in the subjects without cryoglobulinemia, all the other genotypes could be found. An interferon-resistant genotype characterized by an elevated homology with Simmonds' type 2c (rare genotype) was present.