Treatment Outcomes and Predictors of Response in Treatment-Naive HCV Patients Treated with Peginterferon Alfa/Ribavirin in Real-World Italian Clinics: Sub-Analysis from the PROPHESYS Cohort.

BACKGROUND/AIMS: Within Europe, Italy has the highest incidence of HCV. PROPHESYS was a large, non-interventional, multinational cohort study of patients treated with peginterferon alfa-2a or -2b/ribavirin for CHC; 22.4% of patients were from Italian centers. This sub-analysis evaluates real-life practice and treatment outcomes in Italy. METHODOLOGY: The PROPHESYS 2 cohort included 1604 HCV mono-infected, treatment-naive patients. All patients were prescribed peginterferon alfa/ribavirin at the discretion of the treating physician according to country-specific requirements. RESULTS: The majority of G1-3 patients were White/Caucasian and 48.4% had HCV G1 infection. Overall, SVR24 rates of 44.9%, 81.4% and 69.1% were achieved in G1-, 2- and 3-infected patients. In G1 patients, SVR24 rates declined with increasing FIB-4 score; this trend was not observed for G2/3-infected patients. Virologic response by Week 2 and 4 was highly predictive of SVR24 (G1: 91.7%, 84.8%; G2: 91.1%, 89.7%; G3: 92.9%, 86.7%, respectively). Absence of virologic response by Week 12 had the highest negative predictive value across genotypes. CONCLUSIONS: In Italian patients, a virologic response by Week 2 or 4 was highly predictive of SVR24 across genotypes. These data demonstrate the importance of monitoring on-treatment response to help guide treatment decisions. FIB-4 score correlated well with SVR24 in G1 patients.

Activation of VPAC1 receptors aggravates early atherosclerosis in hypercholesterolemic apolipoprotein E-deficient mice.

OBJECTIVE: Vasoactive intestinal peptide (VIP) is a 28-amino acid peptide widely expressed in the body and binding three types of receptors: VPAC(1)-R, VPAC(2)-R and PAC(1)-R. Based on beneficial effects of VIP and VPAC(1)-R agonists in mouse models of several chronic inflammatory disorders, we hypothesized that activation of VIP receptors would prevent atherosclerosis development in apolipoprotein E-deficient mice. METHODS AND RESULTS: Contrary to our hypothesis, administration of a VPAC(1)-R agonist, (Ala(11,22,28))-VIP aggravated atherosclerotic lesion development in the aortic root of these mice compared to control mice. This was accompanied by a significant increase in the expression of MHC class II protein I-A(b), and suggests enhanced inflammatory activity in the vessel wall. The amount of macrophage-specific CD68 staining as well as serum cholesterol and triglyceride levels did not change as a result of the (Ala(11,22,28))-VIP treatment, i.e. the treatment resulted in significant changes in lipid accumulation in the lesions without changing the number of macrophages or systemic lipid levels. Interestingly, administration of VIP did not alter the course of the disease. CONCLUSION: Despite beneficial effects in murine models of several inflammatory disorders, VPAC(1)-R activation aggravates atherosclerotic lesion formation in apolipoprotein E-deficient mice through enhanced inflammatory activity in the vessel wall.

Inhibition of nuclear factor-kappaB activation by IRFI 042, protects against endotoxin-induced shock.

BACKGROUND: The aim of our study was to investigate the effect of IRFI 042, a novel dual vitamin E-like antioxidant, on nuclear factor-kappaB (NF-kappaB) activation, TNF-alpha gene priming and on the release of the mature protein during endotoxin shock. METHODS: Endotoxin shock was produced in male rats by a single intravenous (i.v.) injection of 20 mg kg(-1) of Salmonella enteritidis lipopolysaccharide (LPS). Survival rate, mean arterial blood pressure, serum TNF-alpha and plasma malondialdehyde (MAL) levels were investigated. We then evaluated in the liver TNF-alpha mRNA levels, NF-kappaB binding activity and the inhibitory protein IkappaBalpha. Moreover we studied in LPS stimulated (50 microg ml(-1)) peritoneal macrophages (Mphi), NF-kappaB activation, cytoplasmic IkappaB-alpha degradation, the message for TNF-alpha, and TNF-alpha and MAL levels. RESULTS: LPS administration reduced survival rate (0%, 72 h after LPS administration), decreased mean arterial blood pressure, augmented serum TNF-alpha (60+/-11 ng ml(-1)) and enhanced plasma malondialdehyde (MAL) levels (55+/-7.1 nmol l(-1)). LPS shocked rats also had increased TNF-alpha mRNA levels, augmented liver NF-kappaB binding activity in the nucleus and decreased levels of the inhibitory protein IkappaBalpha. In addition, in vitro LPS stimulation (50 microg ml(-1)) significantly induced NF-kappaB activation and cytoplasmic IkappaBalpha degradation in Mphi, enhanced TNF-alpha mRNA levels and increased Mphi TNF-alpha and MAL. Treatment with IRFI 042 (20 mg kg(-1), i.v., 5 min after endotoxin challenge) protected against LPS-induced lethality (90% survival rate 24 h and 80% survival rate 72 h after LPS injection, respectively), reduced hypotension, blunted plasma MAL (9.0+/-0.9 nmol l(-1)) and decreased serum TNF-alpha (15+/-3 ng ml(-1)). The antioxidant also inhibited the loss of IkappaBalpha protein from the hepatic cytoplasm, blunted the increased NF-kappaB binding activity in the liver and decreased hepatic liver mRNA for TNF-alpha. Furthermore 'in vitro' IRFI 042 (50 microM) significantly inhibited activation of NF-kappaB through inhibition of IkappaBalpha degradation, reduced the amount of TNF-alpha mRNA, decreased LPS-induced TNF-alpha release and blunted lipid peroxidation (MAL) in LPS stimulated Mphi. CONCLUSIONS: These data suggest that IRFI 042 blocks the activation of NF-kappaB, reduces TNF-alpha mRNA levels, and finally reverses endotoxic shock.

Crucial role of nuclear factor-kappaB in neointimal hyperplasia of the mouse carotid artery after interruption of blood flow.

We used a molecular genetics approach to investigate the role of nuclear factor-kappaB (NF-kappaB) in neointimal hyperplasia induced by flow interruption of carotid artery in mice. Wild type mice (WT mice) and mice rendered deficient in p105, the precursor of p50, one of the components of the multimeric transcription factor NF-kappaB (NF-kappaB knockout mice; KO mice), were subjected to a complete ligation of the left common carotid artery. Morphometric analysis of the structural alteration caused by the disruption of the arterial blood flow was performed 14 days after surgery. Furthermore the expression of intercellular adhesion molecule-1 (ICAM-1) in injured arteries was evaluated 4 days after artery ligation by the means of reverse transcriptase polymerase chain reaction (RT-PCR) and quantification of the ICAM-1 protein levels. In a separate experiment normal mice were randomly assigned to receive a recombinant adeno-associated virus (rAAV) encoding the gene for the NF-kappaB inhibitory protein IkappaBalpha (rAAV-IkappaBalpha), or the beta-galactosidase gene (rAAV-LacZ), both at a dose of 10(11) copies and 2 weeks later were subjected to the complete ligation of the left carotid artery. NF-kappaB activity (studied by means of electrophoretic mobility shift assay-EMSA), IkappaBalpha expression (evaluated by Western blot analysis) ICAM-1 evaluation (RT-PCR and quantification of the protein levels) and a morphometric analysis were evaluated in the injured arteries. Disruption of the arterial blood flow caused a marked neointimal hyperplasia. The mean intimal area was 0.023+/-0.002 mm(2) in wild type mice compared with 0.002+/-0.001 mm(2) in NF-kappaB knockout mice. ICAM-1 expression was 1.7+/-0.8 relative amount of ICAM-1 mRNA in wild type mice compared with 0.4+/-0.06 relative amount of ICAM-1 mRNA in NF-kappaB knockout mice. ICAM-1 protein levels were also significantly reduced in NF-kappaB knockout mice. Injured arteries treated with rAAV-IkappaBalpha had a greater expression of IkappaBalpha and lower NF-kappaB activity, when compared with vessels treated with rAAV-LacZ. Furthermore, ICAM-1 expression was markedly attenuated by the treatment with rAAV-IkappaBalpha (rAAV-LacZ=1.6+/-0.8 relative amount of ICAM-1 mRNA; rAAV-IkappaBalpha=0.55+/-0.04 relative amount of ICAM-1 mRNA). ICAM-1 protein levels were also significantly decreased in rAAV-IkappaBalpha treated mice. Finally the mean intimal area was 0.028+/-0.003 mm(2) in left carotid arteries treated with rAAV-LacZ whereas it was 0.003+/-0.004 mm(2) in vessels treated with rAAV-IkappaBalpha. Our data indicate that NF-kappaB plays a crucial role in neointimal hyperplasia induced by flow cessation in the mouse carotid artery, and in addition suggest that rAAV-mediated gene transfer of IkappaBalpha might represent a novel therapeutic approach to the treatment of restenosis.

Real-world outcomes in patients with chronic hepatitis C: primary results of the PROBE study.

BACKGROUND: This large prospective multicentre cohort study aimed to improve knowledge of therapy for chronic hepatitis C (CHC) in real clinical practice. METHODS: A diverse population of adults with CHC including patients with comorbid conditions, laboratory abnormalities and demographic features [comorbidities or special populations (CSP)] who were under-represented or excluded from peginterferon registration studies was treated with peginterferon α-2a (40 kDa) or α-2b (12 kDa) plus ribavirin at the investigator's discretion. RESULTS: During the study, 5399 treatment-naive patients [2527 (46.8%) with CSP] received peginterferon α-2a (n=3513, 65.1%) or peginterferon α-2b (n=1886, 34.9%). The sustained virological response rate was 56.6% (3057/5399) overall, 59.7% (1716/2872) in patients without CSP and 53.1% (1341/2527) in patients with CSP. Significant predictors of sustained virological response included hepatitis C virus genotype 2 or 3 infection, absence of cirrhosis, hepatitis C virus RNA≤500 000 IU/ml, alanine transaminase quotient >3× the upper limit of normal, age ≤65 years, BMI<25 kg/m, at least 80% of the planned exposure to peginterferon and ribavirin and prescription of peginterferon α-2a. CONCLUSION: The results provide detailed information on the outcome of therapy for CHC in a diverse Italian population that included a large number of patients with CSP and provides an insight into the generalizability of the results obtained in the more restricted setting of randomized registration trials.

Effect of recombinant adeno-associated virus vector-mediated vascular endothelial growth factor gene transfer on wound healing after burn injury.

OBJECTIVE: The purpose of this study was to investigate the effect of recombinant adeno-associated viral (rAAV) vector-mediated human vascular endothelial growth factor (VEGF165) transfer on experimental burn wounds. DESIGN: Randomized experiment. SETTING: Research laboratory. SUBJECTS: C57BL/6 male mice weighing 25-30 g. INTERVENTIONS: Mice were immersed in 80 degrees C water for 10 secs to achieve a partial-thickness scald burn. Animals were randomized to receive at two injection sites on the edge of the burn either 1011 copies of the rAAV-VEGF165 or the vector carrying the control and inert gene beta-galactosidase (rAAV-LacZ). On day 14 the animals were killed. Burn areas were used for histologic examination, evaluation of VEGF expression (immunohistochemistry) and VEGF wound content (enzyme-linked immunosorbent assay), determination of wound nitrite, and measurement of messenger RNA (mRNA) for endothelial and inducible nitric oxide synthase (eNOS and iNOS). MEASUREMENTS AND MAIN RESULTS: rAAV-VEGF165 increased epithelial proliferation, angiogenesis, and maturation of the extracellular matrix. Furthermore, gene transfer enhanced VEGF expression, studied by immunohistochemistry, and the wound content of the mature protein (rAAV-LacZ, 11 +/- 5 pg/wound; rAAV-VEGF165, 104 +/- 7 pg/wound). Moreover, VEGF165 gene transfer increased wound content of nitrate. Finally, rAAV-VEGF165 administration enhanced the messenger RNA for eNOS (rAAV-VEGF165, 1.1 +/- 0.2 relative amount of eNOS mRNA; rAAV-LacZ, 0.66 +/- 0.3 relative amount of eNOS mRNA) and iNOS (rAAV-VEGF165, 0.8 +/- 0.09 relative amount of iNOS mRNA; rAAV-LacZ, 0.45 +/- 0.05 relative amount of iNOS mRNA). CONCLUSION: Our study suggests that rAAV-VEGF gene transfer may be an effective therapeutic approach to improve clinical outcomes after thermal injury.

Gene transfer of IkappaBalpha limits infarct size in a mouse model of myocardial ischemia-reperfusion injury.

Nuclear factor-kappaB (NF-kappaB) plays a central role in myocardial ischemia-reperfusion (MI/R) injury. The inhibitory protein IkappaBalpha prevents its activation. We investigated the effects of adeno-associated viral vector-mediated IkappaBalpha gene transfer in MI/R injury. Male C57BL/6 mice were randomized to receive a recombinant adeno-associated virus (rAAV) encoding the gene for the NF-kappaB inhibitory protein IkappaBalpha (rAAV- IkappaBalpha) or the beta-galactosidase gene (a control and inert gene; rAAV-LacZ), both at a dose of 10(11) copies. Four weeks later anesthetized animals were subjected to total occlusion (45 minutes) of the left main coronary artery followed by 5 hours of reperfusion. MI/R produced a wide infarct size (IF/area-at-risk = 56 +/- 8%; IF/left ventricle = 44 +/- 5%) and tissue neutrophil infiltration, studied by means of elastase activity (area-at-risk = 2.5 +/- 0.4 micro g/gm tissue; infarct area = 2.9 +/- 0.6 micro g/gm tissue). Furthermore MI/R caused peak message for intercellular adhesion molecule-1 (ICAM-1) in the area-at-risk at 3 hours of reperfusion (1.2 +/- 0.4 relative amount of cardiac ICAM-1 mRNA). NF-kappaB activation was evident at 0.5 hours of reperfusion and reached its maximum increase at 2 hours of reperfusion. rAAV-IkappaBalpha injection reduced infarct size (IF/area-at-risk = 19 +/- 3%; IF/left ventricle = 10 +/- 2%; p < 0.001), blocked NF-kappaB activation, diminished cardiac ICAM-1 expression (0.4 +/- 0.02 relative amount of cardiac ICAM-1 mRNA; p < 0.001), and blunted leukocyte accumulation (area-at-risk = 0.6 +/- 0.05 micro g/gm tissue; infarct area = 0.4 +/- 0.02 micro g/gm tissue; p < 0.001). Our data indicate that rAAV-IkappaBalpha may be useful for MI/R gene therapy.

Enhancement of expression of vascular endothelial growth factor after adeno-associated virus gene transfer is associated with improvement of brain ischemia injury in the gerbil.

Angiogenesis induced by growth factors may represent a rational therapy for patients with stroke. Vascular endothelial growth factor (VEGF) plays a pivotal role in angiogenesis and VEGF expression is enhanced in the post-ischemic brain. VEGF induced by brain hypoxia can lead to the growth of new vessels and may represent a natural protective mechanism improving survival after stroke. In the light of these findings we investigated changes of VEGF expression in different brain regions after intracerebroventricular injection of adeno-associated virus transferring gene for VEGF (rAAV-VEGF) in the gerbil, and after transient brain ischemic injury, we studied the effects of rAAV-VEGF injection on survival, brain edema, delayed neuronal death in the CA1 area and learning ability. Treatment with rAAV-VEGF 6 days or 12 days before ischemia significantly improves survival, brain edema and CA1 delayed neuronal death and post-ischemic learning evaluated by passive avoidance test. Animals treated with rAAV-VEGF showed in the thalamus and the cortex, a significant positive immunostaining for VEGF similar to those subjected to brain ischemia and not treated with rAAV-VEGF. These data represent a further contribution to a possible employment of gene therapy by using rAAV-VEGF in brain ischemia and indicate that thalamus and cortex may be targets for neuroprotective effects of VEGF.