María de Luján Alvarez

Nitric oxide release and enhancement of lipid peroxidation in regenerating rat liver.

BACKGROUND/AIMS Clarification of the role of lipid peroxidation in the onset of liver proliferation has been hampered by the fact that both higher and lower lipid peroxidation have been reported after two-thirds partial hepatectomy. Recently, it has been shown that nitric oxide might be involved in the control of early responses after partial hepatectomy. We analysed the possible involvement of nitric oxide production in lipid peroxidation levels during liver regeneration. METHODS Sham-operated, hepatectomised and sham and hepatectomised rats pretreated with two inhibitors of oxide nitric synthesis (aminoguanidine or N(G)-monomethyl-L-arginine) were used throughout. Animals were killed at 1, 3, 5 and 15 h after surgery. Cytosolic superoxide dismutase and microsomal-lysosomal catalase activities were measured. Lipid peroxidation levels were measured as thiobarbituric acid-reactive substances and conjugated dienes. Cytosolic nitrate (a stable metabolic product of nitric oxide) was enzymatically determined. Inducible-type nitric oxide synthase (iNOS) was analysed in hepatic cytosol by immunoblotting. DNA synthesis 24 and 48 h after surgery was assessed by [3H]thymidine incorporation. RESULTS Increased lipid peroxidation was found in total homogenate, cytosol and microsomes. The hepatic cytosolic content of nitrates increased, reaching the highest values at 5 h posthepatectomy. Aminoguanidine or N(G)-monomethyl-L-arginine pretreatment blocked the rise of nitric oxide production and lipid peroxidation levels and decreased the DNA synthesis. The increase in hepatic iNOS protein expression at 5 h after partial hepatectomy disappeared with aminoguanidine pretreatment. CONCLUSIONS Our experiments suggest that nitric oxide plays a role in the proliferation mechanism, although it is responsible, at least in part, for the enhanced lipid peroxidation.

Long-term effect of sustained virological response on hepatocellular carcinoma in patients with hepatitis C in Canada

BACKGROUND & AIMS Evidence is limited on hepatocellular carcinoma (HCC) risk after sustained virological response (SVR) to interferon-based treatment of hepatitis C virus (HCV) infection. We evaluated the effect of SVR on the risk of HCC and estimated its incidence in post-SVR HCV patients from a large population-based Canadian cohort. METHODS The British Columbia Hepatitis Testers Cohort includes individuals tested for HCV between 1990-2013 linked with data on their medical visits, hospitalizations, cancers, prescription drugs and mortality. Patients receiving interferon-based HCV treatments were followed from the end of treatment to HCC diagnosis, death or December 31, 2012. We examined HCC risk among those who did and did not achieve SVR using multivariable proportional hazard models with the Fine and Gray modification for competing risks. RESULTS Of 8147 individuals who received HCV treatment and were eligible for analysis, 4663 (57%) achieved SVR and 3484 (43%) did not. Each group was followed for a median of 5.6years (range: 0.5-12.9) for an HCC incidence rate of 1.1/1000 person-years (PY) among the SVR and 7.2/1000 PY among the no SVR group. The HCC incidence rate was higher among those with cirrhosis (SVR: 6.4, no SVR: 21.0/1000 PY). In the multivariable model, SVR was associated with a lower HCC risk (subdistribution hazard ratio [SHR]=0.20, 95% CI: 0.13-0.3), while cirrhosis (SHR=2.61, 95% CI: 1.68-4.04), age ⩾50years, being male and genotype 3 infection were associated with a higher HCC risk. Among those who achieved SVR, cirrhosis, age ⩾50years and being male were associated with a higher HCC risk. CONCLUSION SVR after interferon-based treatment substantially reduces but does not eliminate HCC risk, which is markedly higher among those with cirrhosis and age ⩾50years at treatment initiation. Treatment of patients at an advanced fibrosis stage with new highly effective drugs will warrant continued surveillance for HCC post-SVR. LAY SUMMARY We assessed the effect of successful hepatitis C treatment with older interferon-based treatment on the occurrence of liver cancer (hepatocellular carcinoma) and found that successful treatment prevents liver cancer. However, more people with cirrhosis and older age continued to develop liver cancer after successful treatment. Thus, treatment with new drugs among those with cirrhosis will require continued monitoring for liver cancer.

HCV co-infection in HIV positive population in British Columbia, Canada

BackgroundAs HIV and hepatitis C (HCV) share some modes of transmission co-infection is not uncommon. This study used a population-based sample of HIV and HCV tested individuals to determine the prevalence of HIV/HCV co-infection, the sequence of virus diagnoses, and demographic and associated risk factors.MethodsPositive cases of HIV were linked to the combined laboratory database (of negative and positive HCV antibody results) and HCV reported cases in British Columbia (BC).ResultsOf 4,598 HIV cases with personal identifiers, 3,219 (70%) were linked to the combined HCV database, 1,700 (53%) of these were anti-HCV positive. HCV was diagnosed first in 52% of co-infected cases (median time to HIV identification 3 1/2 years). HIV and HCV was diagnosed within a two week window in 26% of cases. Among individuals who were diagnosed with HIV infection at baseline, subsequent diagnoses of HCV infection was independently associated with: i) intravenous drug use (IDU) in males and females, Hazard Ratio (HR) = 6.64 (95% CI: 4.86-9.07) and 9.76 (95% CI: 5.76-16.54) respectively; ii) reported Aboriginal ethnicity in females HR = 2.09 (95% CI: 1.34-3.27) and iii) males not identified as men-who-have-sex-with-men (MSM), HR = 2.99 (95% CI: 2.09-4.27).Identification of HCV first compared to HIV first was independently associated with IDU in males and females OR = 2.83 (95% CI: 1.84-4.37) and 2.25 (95% CI: 1.15-4.39) respectively, but not Aboriginal ethnicity or MSM. HIV was identified first in 22%, with median time to HCV identification of 15 months;ConclusionThe ability to link BC public health and laboratory HIV and HCV information provided a unique opportunity to explore demographic and risk factors associated with HIV/HCV co-infection. Over half of persons with HIV infection who were tested for HCV were anti-HCV positive; half of these had HCV diagnosed first with HIV identification a median 3.5 years later. This highlights the importance of public health follow-up and harm reduction measures for people identified with HCV to prevent subsequent HIV infection.

Capacity enhancement of hepatitis C virus treatment through integrated, community-based care

BACKGROUND An estimated 250,000 Canadians are infected with the hepatitis C virus (HCV). The present study describes a cohort of individuals with HCV referred to community-based, integrated prevention and care projects developed in British Columbia. Treatment outcomes are reported for a subset of individuals undergoing antiviral therapy at four project sites. METHODS Four demonstration projects based on a public health nurse and physician partnership were established in rural and small urban centres in British Columbia. Comprehensive medical assessments determined whether individuals received treatment, or counselling and education. Outcomes of the treatment group were compared with published randomized controlled trials. Client demographics were mapped using geographical information systems applications. RESULTS A total of 1795 individuals were referred to the clinics for medical assessment between September 2001 and December 2005. After assessment, 26% were eligible for therapy, while 74% received counselling and education. Wait times decreased annually, with one-half of all referrals assessed within 30 days. Combination antiviral therapy was initiated in 363 clients with interferon plus ribavirin (n=36) or pegylated interferon plus ribavirin (n=327). Treatment outcomes were available for 205 individuals. The overall rate of sustained virological response was 61% (126 of 205 individuals). The number of individuals assessed at each site represented, on average, 20% of the total cumulative reported HCV cases in the catchment areas. DISCUSSION The study findings illustrate how a public health nurse and physician partnership can service a population with complex medical needs while simultaneously increasing local capacity. Treatment outcomes were comparable with published clinical trials.

Interferon α-induced apoptosis on rat preneoplastic liver is mediated by hepatocytic transforming growth factor β1

In previous work we showed that interferon alfa‐2b (IFN‐α2b) increases apoptosis on rat hepatic preneoplastic foci. The aim of this study was to determine if transforming growth factor β1 (TGF‐β1) was involved in the programmed cell death on the foci. Animals were divided into 6 groups: subjected to a 2‐phase model (diethylnitrosamine plus 2‐acetylaminofluorene) of preneoplasia development (group 1); treated with IFN‐α2b during the 2 phases (group 2); treated with IFN‐α2b during initiation with diethylnitrosamine (group 3); treated with IFN‐α2b during 2‐acetylaminofluorene administration (group 4); subjected only to an initiation stage (group 5); and treated with IFN‐α2b during the initiation period (group 6). Serum TGF‐β1 levels were increased in IFN‐α2b–treated rats. Immunohistochemical studies showed that IFN‐α2b significantly increased the quantity of TGF‐β1–positive hepatocytes in groups 2 to 4. Phosphorylated‐Smads‐2/3 (p‐Smads‐2/3) proteins in liver nuclear extracts were significantly elevated. To determine the source of TGF‐β1, isolated hepatocytes, Kupffer cells, and peritoneal macrophages from animals in groups 1 and 5 were cultured with or without IFN‐α2b. IFN‐α2b stimulus induced several‐fold increases of TGF‐β1 secretion from hepatocytes. Neither Kupffer cells nor peritoneal macrophages secreted detectable TGF‐β1 levels when they were treated with IFN‐α2b. IFN‐α2b–stimulated cultured hepatocytes from preneoplastic livers showed enhanced apoptosis, measured by fluorescence microscopy and caspase‐3 activity. They presented higher nuclear accumulation of p‐Smads‐2/3, indicating increased TGF‐β1 signaling. When anti–TGF‐β1 was added to the culture media, TGF‐β1 activation and apoptosis induced by IFN‐α2b were blocked. In conclusion, IFN‐α2b–induced production of TGF‐β1 by hepatocytes from preneoplastic liver is involved in the apoptotic elimination of altered hepatic foci. (HEPATOLOGY 2004;40:394–402.)

Quercetin prevents liver carcinogenesis by inducing cell cycle arrest, decreasing cell proliferation and enhancing apoptosis

SCOPE Quercetin is the most abundant flavonoid in human diet. It has special interest as it holds anticancerous properties. This study aims to clarify the mechanisms involved in quercetin effects during the occurrence of preneoplastic lesions in rat liver. METHODS AND RESULTS Adult male Wistar rats were subjected to a two-phase model of hepatocarcinogenesis (initiated-promoted group). Initiated-promoted animals also received quercetin 10 and 20 mg/kg body weight (IPQ10 and IPQ20 groups, respectively). Antioxidant defenses were modified by quercetin administration at both doses. However, only IPQ20 group showed a reduction in number and volume of preneoplastic lesions. This group showed increased apoptosis and a reduction in the proliferative index. In addition, IPQ20 group displayed a reduction of cell percentages in G₁ and S phases, accumulation in G₂, and decrease in M phase, with reduced expression of cyclin D1, cyclin A, cyclin B, and cyclin-dependent kinase 1. Interestingly, peroxisome proliferator activated receptor-α levels were reduced in IPQ20 group. CONCLUSION The outcomes of this study represent a significant contribution to the current understanding on the preventive mechanisms of quercetin during the early stages of liver cancer development, demonstrating that in addition to its known proapoptotic characteristics, the flavonoid modulates the expression of critical cell cycle regulators and peroxisome proliferator activated receptor-α activity.

Interferon-α2b and transforming growth factor-β1 treatments on HCC cell lines: Are Wnt/β-catenin pathway and Smads signaling connected in hepatocellular carcinoma?

Wnt/β-catenin pathway is often dysregulated in hepatocellular carcinoma (HCC). Activated β-catenin accumulates in the cytosol and nucleus and forms a nuclear complex with TCF/LEF factors like TCF4. Interferon-α (IFN-α) has recently been recognized to harbor therapeutic potential in prevention and treatment of HCC. Transforming Growth Factor-β1 (TGF-β1) is a mediator of apoptosis, exerting its effects via Smads proteins. One mode of interaction between Wnt/β-catenin and TGF-β1/Smads pathways is the association of Smads with β-catenin/TCF4. In this study we analyzed the effects of IFN-α2b and TGF-β1 treatments on Wnt/β-catenin pathway, Smads proteins levels, β-catenin/TCF4/Smads interaction and proliferation and apoptotic death in HepG2/C3A and Huh7 cell lines. IFN-α2b and TGF-β1 attenuated Wnt/β-catenin signal by decreasing β-catenin and Frizzled7 receptor proteins contents and the interaction of β-catenin with TCF4. Truncated β-catenin form present in C3A cell line also diminished after treatments. Both cytokines declined Smads proteins and their interaction with TCF4. The overall cellular response to cytokines was the decrease in proliferation and increase in apoptotic death. Treatment with Wnt3a, which elevates β-catenin protein levels, also generated the increment of Smads proteins contents when comparing with untreated cells. In conclusion, IFN-α2b and TGF-β1 proved to be effective as modulators of Wnt/β-catenin pathway in HCC cell lines holding both wild-type and truncated β-catenin. Since the inhibition of β-catenin/TCF4/Smads complexes formation may have a critical role in slowing down oncogenesis, IFN-α2b and TGF-β1 could be useful as potential treatments in patients with HCC.

Tumor necrosis factor alpha induced by Trypanosoma cruzi infection mediates inflammation and cell death in the liver of infected mice

Trypanosoma cruzi (T. cruzi) infected C57BL/6 mice developed a progressive fatal disease due to an imbalance in the profile of circulating related compounds accompanying infection like tumor necrosis factor alpha (TNFalpha). TNFalpha has been proposed as an important effector molecule in apoptosis. In this work, we evaluate inflammation and the proteins involved in apoptotic process in liver of infected mice and the role of TNFalpha. C57BL6/mice were infected subcutaneously with 100 viable trypomastigotes of Tulahuén strain of T cruzi. One set of these animals were treated with 375 microg of antihuman TNFalpha blocking antibody. Animals were sacrificed at 14 days post-infection (p.i).The analyses of Bcl-2 family proteins revealed an increase of the pro-apoptotic proteins Bax and tBid in T. cruzi-infected mice. Compared with control animals, cytochrome c release was increased. Apoptosis was also induced in infected mice. Anti-TNFalpha treatment decreases hepatic apoptosis. Our results suggest that T. cruzi infection induces programmed cell death in the host liver by increase of TNFalpha production, associated with TNF-R1 over-expression, that set in motion the Bid cleavage and mitochondrial translocation, Bax mitochondrial translocation, cytochrome c release, and ultimately apoptosis induction.

Twin epidemics of new and prevalent hepatitis C infections in Canada: BC Hepatitis Testers Cohort

BackgroundWe characterized the twin epidemics of new and prevalent hepatitis C virus (HCV) infections in British Columbia, Canada to inform prevention, care and treatment programs.MethodsThe BC Hepatitis Testers Cohort (BC-HTC) includes individuals tested for HCV, HIV or reported as a case of HBV, HCV, HIV or active TB between 1990–2013 linked with data on their medical visits, hospitalizations, cancers, prescription drugs and mortality. Prevalent infection was defined as being anti-HCV positive at first test. Those with a negative test followed by a positive test were considered seroconverters or new infections.ResultsOf 1,132,855 individuals tested for HCV, 64,634 (5.8 %) were positive and an additional 3092 cases tested positive elsewhere for a total of 67,726. Of 55,781 HCV positive individuals alive at the end of 2013, 7064 were seroconverters while 48,717 had prevalent infection at diagnosis. The HCV positivity rate (11.2 %) was highest in birth cohort 1945–1964 which declined over time. New infections were more likely to be male, 15–34 years of age (born 1965-1984), HIV- or HBV-coinfected, socioeconomically disadvantaged, have problematic drug and alcohol use and a mental health illness. The profile was similar for individuals with prevalent infection, except for lower odds of HBV-coinfection, major mental health diagnoses and birth cohort >1975.ConclusionsThe HCV positivity rate is highest in birth cohort 1945–1964 which represents most prevalent infections. New infections occur in younger birth cohorts who are commonly coinfected with HIV and/or HBV, socioeconomically marginalized, and living with mental illness and addictions.

Cross-talk between IFN-α and TGF-β1 signaling pathways in preneoplastic rat liver

Interferon-γ/transforming growth factor-β (IFN-γ/TGF-β) pathways have opposite effects on diverse cellular functions. However, little is known about interactions between IFN-α/TGF-β. In previous studies, we showed that IFN-α2b increases TGF-β1 production and secretion in hepatocytes from preneoplastic rat livers. Here, the interaction between IFN-α/TGF-β1 pathways was explored. We observed a positive cross-talk between IFN-α and TGF-β1 signaling, with activation of both pathways. p300 protein levels in hepatocytes from preneoplastic livers were enough to interact with both activated Stat1 and Smad2/3. Besides, Smad7 was not directly related with TGF-β1 and IFN-α signals. Interestingly, we reported the novel finding that the autocrine TGF-β1 up-regulates TGF-βRII at protein and mRNA levels. In conclusion, the intracellular signals triggered by IFN-α2b and by autocrine TGF-β1 are integrated at the nuclear level, where activated Stat1 and Smad2/3 are capable of interact with p300, present in no restrictive cellular amounts.