Biomedical subjects

M Levrero

Publications and source records attributed to M Levrero.

At least 55 records · Page 3Linked to original sources

PubMed1997

Tumor necrosis factor (TNF) receptor 1 signaling downstream of TNF receptor-associated factor 2. Nuclear factor kappaB (NFkappaB)-inducing kinase requirement for activation of activating protein 1 and NFkappaB but not of c-Jun N-terminal kinase/stress-activated protein kinase.

Like other members of the tumor necrosis factor (TNF) receptor family, p55 TNF receptor 1 (TNF-R1) lacks intrinsic signaling capacity and transduces signals by recruiting associating molecules. The TNF-R1 associated death domain protein interacts with the p55 TNF-R1 cytoplasmic domain and recruits the Fas-associated death domain protein (which directly activates the apoptotic proteases), the protein kinase receptor interacting protein, and TNF receptor-associated factor 2 (TRAF2). TRAF2 has previously been demonstrated to activate both transcription factor nuclear factor kappaB (NFkappaB) and the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway, which in turn stimulates transcription factor activating protein 1 (AP1) mainly via phosphorylation of the c-Jun component. We have investigated the signaling properties of NFkappaB-inducing kinase (NIK), a TRAF2-associated protein kinase that mediates NFkappaB induction. NIK was found to be unable to activate JNK/SAPK, mitogen-activated protein kinase, or p38 kinase. Moreover, NIK was not required for JNK/SAPK activation by TNF-R1, thus representing the first TNF-R1 complex component to dissect the NFkappaB and the JNK/SAPK pathways. Despite being unable to activate JNK/SAPK and mitogen-activated protein kinase, NIK strongly activated AP1 and was required for TNF-R1-induced AP1 activation. Therefore, NIK links TNF-R1 to a novel, JNK/SAPK-independent, AP1 activation pathway.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1997

The hepatitis B virus X gene induces p53-mediated programmed cell death.

The human hepatitis B virus (HBV) protein pX is a multifunctional regulatory protein that is known to affect both transcription and cell growth. Here we describe induction of apoptosis in NIH 3T3 polyclonal cell lines upon stimulation of pX expression from a dexamethasone inducible mouse mammary tumor virus (MMTV)-X expression vector. The effect of long-term pX expression on the cell survival of mouse fibroblasts was confirmed in colony generation assays. This effect is not shared either by the other HBV products and it is c-myc mediated, as shown by the use of a dominant negative deletion mutant of c-myc. pX also sensitize cells to programmed cell death after exposure to DNA damaging agents. Taking advantage of stable transfectants carrying the p53val135 temperature-sensitive allele, we directly demonstrate that induction of apoptosis by pX requires p53. In p53 null mouse embryo fibroblasts pX activates transcription and confers an evident growth advantage without loss of cell viability. Although pX protein was not detectable in the experimental conditions we used, our results indicate that its expression affects both cell growth and cell death control.

3T3 Cells↗

PubMed1997

Uncoupling of p21 induction and MyoD activation results in the failure of irreversible cell cycle arrest in doxorubicin-treated myocytes.

Doxorubicin (Dox, Adriamicin), a potent broad spectrum anthracycline anticancer drug, selectively inhibits muscle specific gene expression in cardiac cells in vivo and prevents terminal differentiation of skeletal muscle cells in vitro. By inducing the expression of the helix-loop-helix (HLH) transcriptional inhibitor ld2, Dox represses the myogenic function of the MyoD family of muscle regulatory factors (MRFs). In many cell types, terminal differentiation is coupled to an irreversible exit from the cell cycle and MyoD plays a critical role in the permanent cell cycle arrest of differentiating myocytes by upregulating the cyclin dependent kinase inhibitor (cdki) p21. Here, we correlate Dox effects on cell cycle with changes of E2F/DP complexes and activity in differentiating C2C12 myocytes. In Dox-treated quiescent myoblasts, which fail to differentiate into myotubes under permissive culture conditions, serum re-stimulation induces cyclin/cdk re-association on the E2F/DP complexes and this correlates with an evident increase in E2F/DP driven transcription and re-entry of myoblasts into the cell cycle. Despite Dox ability to activate the DNA-damage dependent p53/p21 pathway, when induced in the absence of MyoD or other MRFs, p21 fails to maintain the postmitotic state in Dox-treated myocytes induced to differentiate. Thus, uncoupling p21 induction and MyoD activity results in a serum-reversible cell cycle arrest, indicating that MRF specific activation of cdki(s) is required for permanent cell cycle arrest in differentiating muscle cells.

Animals↗

PubMed1997

MyoD prevents cyclinA/cdk2 containing E2F complexes formation in terminally differentiated myocytes.

Withdrawal from the cell cycle of differentiating myocytes is regulated by the myogenic basic helix-loop-helix (bHLH) protein MyoD and the pocket proteins pRb, p107 and pRb2/p130. Downstream effectors of 'pocket' proteins are the components of the E2F family of transcription factors, which regulate the G1/S-phase transition. We analysed by EMSA the composition of E2F complexes in cycling, quiescent undifferentiated and differentiated C2C12 skeletal muscle cells. An E2F complex containing mainly E2F4 and pRb2/p130 (E2F-G0/G1 complex) appears when DNA synthesis arrests, replacing the cyclinA/cdk2 containing E2F complex of proliferating myoblasts (E2F-G1/S complex). Serum stimulation reinduces DNA synthesis and the re-appearance of E2F-G1/S complexes in quiescent myoblasts but not in differentiated C2C12 myotubes. In differentiating C2C12 cells, E2F complexes switch and DNA synthesis in response to serum are prevented when MyoD DNA binding activity and the cdks inhibitor MyoD downstream effector p21 are induced. Thus, during myogenic differentiation, formation of E2F4 and pRb2/p130 containing complexes is an early event, but not enough on its own to prevent the reactivation of DNA synthesis. Using a subclone of C3H10T1/2 mouse fibroblasts stably expressing Estrogen Receptor-MyoD (ER-MyoD) chimerae, we found that estrogen directed MyoD activation prevents the reassociation of cyclinA/cdk2 to the E2F4 containing complex following serum stimulation and this correlates with suppression of E2F activity and the inability of cells to re-enter the cell cycle. Our data indicate that, in differentiating myocytes, one mechanism through which MyoD induces permanent cell cycle arrest involves p21 upregulation and suppression of the proliferation-associated cdks-containing E2F complexes formation.

Animals↗

PubMed1997

p300 is required for MyoD-dependent cell cycle arrest and muscle-specific gene transcription.

The nuclear phosphoprotein p300 is a new member of a family of 'co-activators' (which also includes the CREB binding protein CBP), that directly modulate transcription by interacting with components of the basal transcriptional machinery. Both p300 and CBP are targeted by the adenovirus E1A protein, and binding to p300 is required for E1A to inhibit terminal differentiation in both keratinocytes and myoblasts. Here we demonstrate that, in differentiating skeletal muscle cells, p300 physically interacts with the myogenic basic helix-loop-helix (bHLH) regulatory protein MyoD at its DNA binding sites. During muscle differentiation, MyoD plays a dual role: besides activating muscle-specific transcription, it induces permanent cell cycle arrest by up-regulating the cyclin-dependent kinase inhibitor p21. We show that p300 is involved in both these activities. Indeed, E1A mutants lacking the ability to bind p300 are greatly impaired in the repression of E-box-driven transcription, and p300 overexpression rescues the wild-type E1A-mediated repression. Moreover, p300 potentiates MyoD- and myogenin-dependent activation of transcription from E-box-containing reporter genes. We also provide evidence, obtained by microinjection of anti-p300 antibodies, that p300 is required for MyoD-dependent cell cycle arrest in either myogenic cells induced to differentiate or in MyoD-converted C3H10T1/2 fibroblasts, but is dispensable for maintenance of the postmitotic state of myotubes.

Animals↗

PubMed1997

Activation of SAPK/JNK by TNF receptor 1 through a noncytotoxic TRAF2-dependent pathway.

Interaction of the p55 tumor necrosis factor receptor 1 (TNF-R1)-associated signal transducer TRADD with FADD signals apoptosis, whereas the TNF receptor-associated factor 2 protein (TRAF2) is required for activation of the nuclear transcription factor nuclear factor kappa B. TNF-induced activation of the stress-activated protein kinase (SAPK) was shown to occur through a noncytotoxic TRAF2-dependent pathway. TRAF2 was both sufficient and necessary for activation of SAPK by TNF-R1; conversely, expression of a dominant-negative FADD mutant, which blocks apoptosis, did not interfere with SAPK activation. Therefore, SAPK activation occurs through a pathway that is not required for TNF-R1-induced apoptosis.

Acetylcysteine↗

PubMed1997

Primary effusion Burkitt's lymphoma with t(8;22) in a patient with hepatitis C virus-related cirrhosis.

Hepatitis C virus (HCV) infection may be complicated by non-Hodgkin's lymphoma through yet unknown pathogenetic mechanisms. We describe the case of a patient with HCV-related cirrhosis who developed a primary effusion lymphoma (PEL) of Burkitt's type confined to the peritoneal cavity, in the absence of immunodeficiency or autoimmunity. Paracentesis followed by immunophenotyping, karyotyping, and molecular studies allowed us to diagnose a small noncleaved B-cell lymphoma (CD20+, CD24+, CD10+, CD5-, CD23-, lambda+) with the t(8;22) (q24;q11) translocation and clonal rearrangement of the immunoglobulin heavy chain gene. HCV-RNA, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus were not identified within lymphoma cells. The finding of HCV-RNA in the ascitic fluid suggests a link between HCV and development of lymphoma with HCV playing the role of persistent antigenic stimulation to intraperitoneal B-cell clonal expansion(s).

Ascitic Fluid↗

PubMed1997

Differential roles of p300 and PCAF acetyltransferases in muscle differentiation.

PCAF is a histone acetyltransferase that associates with p300/CBP and competes with E1A for access to them. While exogenous expression of PCAF potentiates both MyoD-directed transcription and myogenic differentiation, PCAF inactivation by anti-PCAF antibody microinjection prevents differentiation. MyoD interacts directly with both p300/CBP and PCAF, forming a multimeric protein complex on the promoter elements. Viral transforming factors that interfere with muscle differentiation disrupt this complex without affecting the MyoD-DNA interaction, indicating functional significance of the complex formation. Exogenous expression of PCAF or p300 promotes p21 expression and terminal cell-cycle arrest. Both of these activities are dependent on the histone acetyltransferase activity of PCAF, but not on that of p300. These results indicate that recruitment of histone acetyltransferase activity of PCAF by MyoD, through p300/CBP, is crucial for activation of the myogenic program.

Acetyl-CoA C-Acyltransferase↗

PubMed1997

Fas/Apo1 mutations and autoimmune lymphoproliferative syndrome in a patient with type 2 autoimmune hepatitis.

Inherited mutations of the Fas/Apo1/CD95 gene, a cell-surface receptor involved in cell death signaling and in the control of self-reactivity, characterize the recently identified autoimmune lymphoproliferative syndromes. A patient with type 2 autoimmune hepatitis with the immunologic and genetic features of autoimmune lymphoproliferative syndrome is described. The clinical picture was dominated by liver disease with hepatosplenomegaly and positivity for anti-liver-kidney microsome 1 and anti-liver-cytosol 1 antibodies. A marked increase in CD3+CD4-CD8-T lymphocytes and inherited mutations in Fas alleles that led to the expression of a soluble form of the protein were also found. Fas-mediated apoptosis was deficient in the patient as it was in her mother and her sister, who carried the same allele 2 mutation. This observation links type 2 autoimmune hepatitis, an organ-specific disease, with a genetically determined defect in peripheral tolerance control.

Alleles↗

PubMed1997

A temperature-conditional mutant of simian virus 40 large T antigen requires serum to inhibit myogenesis and does not induce DNA synthesis in myotubes.

The temperature-conditional mutant tsA58 of SV40 large T antigen (Tag) increases the proliferation rate and the number of cell divisions in primary murine and human myogenic cells when expressed under permissive conditions (i.e., at 33 degrees C in medium containing high levels of serum). Under these conditions, Tag also prevents terminal differentiation. Under nonpermissive conditions (i.e., at 39 degrees C in medium containing low levels of serum) in which Tag is largely inactive, proliferation is arrested, and differentiation occurs. However, even at a permissive temperature, the removal of serum induced myosin expression and the fusion of myogenic cells, which continued to express functional Tag. Although Tag was complexed with pRb, as expected from a functional protein, proliferation was nevertheless arrested, and differentiation was induced. Consistent with these findings, the exposure of Tag-expressing differentiated myotubes to serum at 33 degrees C did not reinduce DNA synthesis in these cells. Thus, in myogenic cells, temperature-conditional mutants of Tag stimulate proliferation in the presence of serum but neither prevent terminal differentiation in the absence of serum nor induce DNA synthesis once complete withdrawal from the cycle has occurred.

Animals↗

PubMed1996

Long-lasting memory T cell responses following self-limited acute hepatitis B.

The molecular and cellular basis of long-term T cell memory against viral antigens is still largely undefined. To characterize anti-viral protection by memory T cells against non-cytopathic viruses able to cause acute self-limited and chronic infections, such as the hepatitis B virus (HBV), we studied HLA class II restricted responses against HBV structural antigens in 17 patients with acute hepatitis B, during the acute stage of infection and 2.2 to 13 yr after clinical resolution of disease. Results indicate that: (a) significant T cell proliferative responses to HBV nucleocapsid antigens were detectable in all patients during the acute phase of infection and in 14/17 also 2-13 yr after clinical resolution of disease; b) long-lasting T cell responses were sustained by CD45RO+T cells, predominantly expressing the phenotype of recently activated cells; c) limiting dilution analysis showed that in some patients the frequency of HBV-specific T cells was comparable to that observed in the acute stage of infection and, usually, higher than in patients with chronic HBV infection; d) the same amino acid sequences were recognized by T cells in the acute and recovery phases of infection; and e) HBV-DNA was detectable by nested-PCR in approximately half of the subjects. to conclusion, our results show that vigorous anti-viral T cell responses are detectable in vitro several years after clinical recovery from acute hepatitis B. Detection of minute amounts of virus in some recovered subjects suggests that long-term maintenance of an active anti-viral T cell response could be important not only for protection against reinfection but also for keeping the persisting virus under tight control.

Acute Disease↗

PubMed1996

Elevated serum levels of 90K/MAC-2 BP predict unresponsiveness to alpha-interferon therapy in chronic HCV hepatitis patients.

BACKGROUND: The clinical outcome of hepatitis virus infections is though to depend on the complex interplay between the host immune response profile and virus factors. 90K/MAC-2 BP is a novel member of the Scavenger Receptor Cysteine Rich protein superfamily that functions as a molecular alarm signal for the cellular immune system against both cancer cells and virus infections. METHODS: To assess the significance and the potential clinical usefulness of testing for serum levels of 90K/MAC-2 BP in chronic viral hepatitis patients we studied 115 consecutive patients with chronic HCV hepatitis, 28 HBsAg chronic hepatitis patients, 12 asymptomatic HCV carriers and 11 asymptomatic HBV carriers. 103 out of the 115 HCV patients have been treated with recombinant alpha 2a-interferon at the dose of 3 Mega Units (MU) t.i.w. for 6 months followed by 1.5 MU t.i.w. for 6 months, and have been followed up for a further 12 months. Serum levels of 90K/MAC-2 BP were measured by an immunoradiometric assay based on the specific SP-2 monoclonal antibody. RESULTS AND CONCLUSIONS: Serum 90K/MAC-2 BP levels are increased in chronic viral hepatitis patients, being significantly higher in HCV than in HBV patients. In chronic HCV hepatitis, serum 90K/MAC-2 BP levels are related to both the degree of disease severity and duration of infection. Moreover, elevated 90K/MAC-2 BP serum levels are an independent predictor of failure to respond to alpha-interferon treatment in a cohort of community-acquired chronic hepatitis C patients.

Adult↗

PubMed1996

Hepatitis B virus pX activates NF-kappa B-dependent transcription through a Raf-independent pathway.

In this study, we characterized the molecular events involved in the activation of the ubiquitous transcription factor NF-kappa B by the viral transactivator pX. pX expression in HeLa cells determines a manyfold increase in NF-kappa B-dependent transcription, which is associated with an increase in p50/p65 heterodimer DNA-binding activity. Since the I kappa B-alpha inhibitory subunit proteolytic degradation, which follows its phosphorylation/modification, is a key event in NF-kappa B activation by different stimuli (such as growth factors, phorbol esters, tumor necrosis factor, UV irradiation, and oxygen radicals), we investigated pX effects on I kappa B-alpha, as well as the possible involvement of known signalling pathways in pX-induced NF-kappa B-dependent transcription. We observed that although pX had no direct effect on p50 or p65, it was able to restore the I kappa B-alpha-suppressed p50/p65 activity. More directly, the stable expression of pX in HeLa cells resulted in reduced levels of I kappa B-alpha in the cytoplasm. Pretreatment of the cells with H7, calphostin C, tyrphostin 25, or N-acetylcysteine did not impair the effects of pX on NF-kappa B, thus ruling out the involvement of protein kinase C, tyrosine kinases, and oxygen radicals. Finally, while most of the known NF-kappa B-activating agents converge on Raf-1 protein kinase, when Raf-1 activity is blocked by overexpression of a dominant negative mutant, the effects of pX on NF-kappa B are not impaired. Thus, we suggest that although pX is able to activate the Ras/Raf-1-signalling pathway, it triggers NF-kappa B activation by an as yet unidentified Raf-1-independent pathway.

Base Sequence↗

PubMed1995

Reactive oxygen intermediates mediate angiotensin II-induced c-Jun.c-Fos heterodimer DNA binding activity and proliferative hypertrophic responses in myogenic cells.

Angiotensin II (Ang-II) receptor engagement activates many immediate early response genes in both vascular smooth muscle cells and cardiomyocytes whether a hyperplastic or hypertrophic response is taking place. Although the signaling pathways stimulated by Ang-II in different cell lines have been widely characterized, the correlation between the generation of different second messengers and specific physiological responses remains relatively unexplored. In this study, we report how in both C2C12 quiescent myoblasts and differentiated myotubes Ang-II significantly stimulates AP1-driven transcription and c-Jun.c-Fos heterodimer DNA binding activity. Using a set of different protein kinase inhibitors, we could demonstrate that Ang-II-induced increase in AP1 binding is not mediated by the cAMP-dependent pathway and that both protein kinase C and tyrosine kinases are involved. The observation that in quiescent myoblasts Ang-II increase of AP1 binding and induction of DNA synthesis and, in differentiated myotubes, Ang-II stimulation of protein synthesis are abolished by the cysteine-derivative and glutathione precursor N-acetyl-L-cysteine strongly suggests a role for reactive oxygen intermediates in the intracellular transduction of Ang-II signals for immediate early gene induction, cell proliferation, and hypertrophic responses.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1995

Resistance to Fas-mediated apoptosis in human hepatoma cells.

CTLs- and lymphokine-induced apoptosis of infected hepatocytes during the course of chronic viral hepatitis is thought to be important for both disease termination and prevention of hepatocellular transformation. We therefore studied apoptosis induced by Fas (APO-1 or CD95)-a widely expressed cell surface receptor whose ligand is involved in lymphocyte cytotoxicity-in a set of human hepatoma cell lines. As normal hepatocytes, all of the human hepatoma cell lines tested do express detectable amounts of Fas on their surface. Nevertheless, only PLC/PRF/5 cells undergo apoptosis following treatment with anti-Fas. Systematic cloning and sequence analysis of the Fas cDNA did not show mutations in the Fas gene in any of the cells lines tested. However, due to alternative splicing, 5 to 10% of the Fas cDNAs are deleted of 63 internal nucleotides corresponding to the transmembrane domain, thus encoding for a soluble and secreted form of Fas (Fas delta TM), potentially able to neutralize anti-Fas or Fas-Ligand. Although we could not demonstrate a direct correlation between resistance of different hepatoma cell lines to Fas mediated death and endogenous expression of this transcript, we show that PLC/PRF 5 stable transfectants overexpressing Fas delta TM are less sensitive to anti-Fas than control cells. In three different cell lines, resistance to anti-Fas was overcome by treatment with the protein synthesis inhibitor cycloheximide. Although this could suggest the existence of short-lived repressors of the Fas-activated apoptotic signalling pathway(s), we show that translational inhibition is not required for the synergistic effect of cycloheximide to take place, and that resistant hepatoma cells can be sensitized to anti-Fas by subinhibitory concentrations of this protein synthesis inhibitor. Since cycloheximide is able to activate intracellular signalling independently on its effects on protein synthesis, we suggest that it might provide a costimulatory signal that cooperates with Fas in the induction of cell death and that, at least in the cells we tested, resistance to Fas is not an active process involving gene transcription and translation but only the consequence of an inadequate apoptotic stimulation.

Adult↗

PubMed1995

Reactive oxygen intermediates (ROIs) are involved in the intracellular transduction of angiotensin II signal in C2C12 cells.

Increasing evidence suggests that angiotensin II may act as a growth factor for several muscle cell types. Angiotensin II stimulation activates many immediate early response genes like c-Fos, c-Jun, c-Myc and Egr-1 in both vascular smooth muscle cells and cardiomyocytes, independently of whether a hyperplastic or hypertrophic response is taking place. In this study we report that angiotensin II significantly stimulates AP1-driven transcription in mouse skeletal muscle cells C2C12 stably transfected with a TRE-tk-CAT plasmid in a dose-dependent manner (peak stimulation at 10(-5) M of angiotensin II). Moreover, angiotensin II increases the binding of the AP1 complex to its DNA target in both quiescent C2C12 myoblasts and in differentiated C2C12 myotubes. Most of the TRE-bound complexes in both unstimulated and angiotensin II-treated cells consist of c-jun/c-fos heterodimers. Using a set of different protein kinase inhibitors, including HA1004, H7, tyrphostin, genistein and staurosporine, we could demonstrate that the angiotensin II-induced AP1 binding increase is not mediated by the cAMP-dependent pathway and that protein kinase C and tyrosine kinases are involved. Treatment of C2C12 cells with H2O2 induces a dose-dependent increase in c-jun/c-fos heterodimer binding, specifically reverted by the cysteine derivative and glutathione precursor N-acetyl-L-cysteine (NAC). The observation that the induction by angiotensin II of both the AP1 DNA binding activity and DNA synthesis in quiescent C2C12 myoblasts is abolished by NAC strongly suggests a role for reactive oxygen intermediates (ROIs) in the intracellular transduction of angiotensin II signals for immediate early gene induction and for cell proliferation.

Angiotensin II↗

PubMed1995

Infection of circulating and liver infiltrating T cells by hepatitis C virus of different subtypes.

Hepatitis C virus (HCV) infection display a very high rate of progression to chronicity and, like many other viruses causing persistent infections, it displays a tropism for the cells of the immune system. Peripheral blood mononuclear cells (PBMCs) from 21 HCV chronic carriers and long-term T cell clones derived from circulating or liver infiltrating T lymphocytes were tested by cDNA "nested" PCR for positive and negative strand HCV-RNA. The presence of HCV genomes in PBMCs is a frequent, although not constant, finding and can be accompanied by active viral replication, as suggested by the coexistence of negative strand HCV-RNA. Infected T cells are more represented in livers than in periphery, as indicated by comparing HCV-RNA detection in T cell clones isolated from both the compartments. Sequencing of viral genomes present in PBMCs and liver infiltrating lymphocytes showed that all the three major HCV genotypes present in our population of chronic carriers can infect lymphoid cells. Although each clonal population of T cells is infected by a single strain of HCV, in the same patient lymphoid cells can harbor different viral populations, different from those circulating at that moment in the serum.

Adult↗

PubMed1995

Distribution of three major hepatitis C virus genotypes in Italy. A multicentre study of 495 patients with chronic hepatitis C.

Different genotypes of hepatitis C virus (HCV) have been shown to have distinct geographical distribution and to associate with variable clinical features. To evaluate their role in chronic hepatitis in Italian patients, we studied 495 consecutive cases with chronic hepatitis C seen in nine sentinel centres homogeneously distributed over Italy. HCV genotyping was carried out using a dot-blot hybridization assay with genotype-specific probes. Four hundred and eleven patients were viraemic and could be evaluated: 57% were found to be infected with HCV-1, 31% with HCV-2, 8% with HCV-3, 1% showed mixed infection and 3% were ascribed to HCV-2b or HCV-4 by direct sequencing. Geographical distribution showed discrete territorial variations. A history of drug addiction was commoner in patients infected with HCV-3. There were no significant differences in activity of liver disease among different HCV genotypes but the response to interferon therapy was reduced in patients infected with HCV-1 compared to HCV-2 or HCV-3.

Adult↗