Sorafenib prevents liver fibrosis in a non-alcoholic steatohepatitis (NASH) rodent model

Liver fibrosis occurring as an outcome of non-alcoholic steatohepatitis (NASH) can precede the development of cirrhosis. We investigated the effects of sorafenib in preventing liver fibrosis in a rodent model of NASH. Adult Sprague-Dawley rats were fed a choline-deficient high-fat diet and exposed to diethylnitrosamine for 6 weeks. The NASH group (n=10) received vehicle and the sorafenib group (n=10) received 2.5 mg·kg-1·day-1 by gavage. A control group (n=4) received only standard diet and vehicle. Following treatment, animals were sacrificed and liver tissue was collected for histologic examination, mRNA isolation, and analysis of mitochondrial function. Genes related to fibrosis (MMP9, TIMP1, TIMP2), oxidative stress (HSP60, HSP90, GST), and mitochondrial biogenesis (PGC1α) were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR). Liver mitochondrial oxidation activity was measured by a polarographic method, and cytokines by enzyme-linked immunosorbent assay (ELISA). Sorafenib treatment restored mitochondrial function and reduced collagen deposition by nearly 63% compared to the NASH group. Sorafenib upregulated PGC1α and MMP9 and reduced TIMP1 and TIMP2 mRNA and IL-6 and IL-10 protein expression. There were no differences in HSP60, HSP90 and GST expression. Sorafenib modulated PGC1α expression, improved mitochondrial respiration and prevented collagen deposition. It may, therefore, be useful in the treatment of liver fibrosis in NASH.

MTP -493G/T gene polymorphism is associated with steatosis in hepatitis C-infected patients

The reduction of hepatic microsomal transfer protein (MTP) activity results in fatty liver, worsening hepatic steatosis and fibrosis in chronic hepatitis C (CHC). The G allele of the MTP gene promoter, -493G/T, has been associated with lower transcriptional activity than the T allele. We investigated this association with metabolic and histological variables in patients with CHC. A total of 174 untreated patients with CHC were genotyped for MTP -493G/T by direct sequencing using PCR. All patients were negative for markers of Wilson’s disease, hemochromatosis and autoimmune diseases and had current and past daily alcohol intake lower than 100 g/week. The sample distribution was in Hardy-Weinberg equilibrium. Among subjects with genotype 1, 56.8 percent of the patients with fibrosis grade 3+4 presented at least one G allele versus 34.3 percent of the patients with fibrosis grade 1+2 (OR = 1.8; 95 percentCI = 1.3-2.3). Logistic regression analysis with steatosis as the dependent variable identified genotypes GG+GT as independent protective factors against steatosis (OR = 0.4, 95 percentCI = 0.2-0.8; P = 0.01). The results suggest that the presence of the G allele of MTP -493G/T associated with lower hepatic MTP expression protects against steatosis in our CHC patients.

Níveis de fósforo, cobre, cobalto e zinco em bubalinos (Bubalus bubalis) na Ilha de Marajó, Estado do Pará / Levels of phosphorus, copper, cobalt and zinc in water buffaloes (Bubalus bubalis) on Marajo Island, Pará

Foram estudados 104 bubalinos, adultos, sem distinção de raça e sexo, criados extensivamente, sem suplementação, em pastagens nativas de baixa qualidade nutricional, dos municípios de Breves, Cachoeira do Arari, Salvaterra e Soure, Ilha de Marajó, Pará. Realizou-se coleta de amostras de fígado, osso e sangue de 26 animais do município de Salvaterra, 38 animais do município de Soure, 20 animais do município de Breves e 20 animais do município de Cachoeira do Arari. Foram realizadas determinações dos teores de fósforo, no soro sanguíneo e no osso, do percentual de cinzas e da densidade específica no tecido ósseo, e de cobalto, cobre e zinco no tecido hepático. Observou-se que a média das concentrações de fósforo no soro sanguíneo (6,26mg/dl) e no osso (10,77%), a percentagem de cinzas (60,87%) e a densidade específica (1,59g/ml) do osso foram inferiores aos níveis críticos estabelecidos para bovinos, caracterizando deficiência de fósforo. As concentrações médias de cobre (5,57ppm), e zinco (27,05ppm) foram consideradas baixas quando comparadas com valores de referência, caracterizando deficiência para estes elementos. No caso do cobalto, quando se considerou os valores detectáveis e não detectáveis pela metodologia observou-se que 51,92% dos animais apresentaram níveis inferiores aos de referência, demonstrando a ocorrência da deficiência de cobalto nesses animais. Ressalta-se que as deficiências de cobre e zinco demonstraram uma maior gravidade já que todos os animais estudados apresentaram níveis deficientes nesses elementos.

The study comprised 104 adults buffaloes, without distinction of race and sex, reared extensively, without supplementation, in native pastures of low nutritional quality, in the municipalities of Breves, Cachoeira do Arari, Salvaterra and Soure, Marajo Island, Pará. Samples of liver, bone and blood were collected from 26 animals in the municipality of Salvaterra, from 38 animals in the municipality of Soure, from 20 animals in the municipality of Breves and from 20 animals in the municipality of Cachoeira do Arari. Determination of the levels of phosphorus in the blood serum and bone ash, and of the specific gravity of bone, and of cobalt, copper and zinc in liver tissue were performed. The average phosphorus concentrations in blood serum (6.26mg/dl) and bone (10.77%), the percentage of ash (60.87%) and specific gravity (1.59g/ml) of bone were lower than the critical levels established for cattle, characterizing phosphorus deficiency. The average concentrations of copper (5.57ppm) and zinc (27.05ppm) was considered low when compared with reference values, characterizing deficiency for these elements. In the case of cobalt when considering the detectable and undetectable values by the methodology, it was observed that 51.92% of the animals had levels below the reference, indicating the occurrence of cobalt deficiency in these animals. It is emphasized that the deficiencies of copper and zinc were the most severe since all animals studied showed deficient levels of these elements.

Differential gene expression profiles of hepatocellular carcinomas associated or not with viral infection

Chronic hepatitis B (HBV) and C (HCV) virus infections are the most important factors associated with hepatocellular carcinoma (HCC), but tumor prognosis remains poor due to the lack of diagnostic biomarkers. In order to identify novel diagnostic markers and therapeutic targets, the gene expression profile associated with viral and non-viral HCC was assessed in 9 tumor samples by oligo-microarrays. The differentially expressed genes were examined using a z-score and KEGG pathway for the search of ontological biological processes. We selected a non-redundant set of 15 genes with the lowest P value for clustering samples into three groups using the non-supervised algorithm k-means. Fisher’s linear discriminant analysis was then applied in an exhaustive search of trios of genes that could be used to build classifiers for class distinction. Different transcriptional levels of genes were identified in HCC of different etiologies and from different HCC samples. When comparing HBV-HCC vs HCV-HCC, HBV-HCC/HCV-HCC vs non-viral (NV)-HCC, HBC-HCC vs NV-HCC, and HCV-HCC vs NV-HCC of the 58 non-redundant differentially expressed genes, only 6 genes (IKBKâ, CREBBP, WNT10B, PRDX6, ITGAV, and IFNAR1) were found to be associated with hepatic carcinogenesis. By combining trios, classifiers could be generated, which correctly classified 100 percent of the samples. This expression profiling may provide a useful tool for research into the pathophysiology of HCC. A detailed understanding of how these distinct genes are involved in molecular pathways is of fundamental importance to the development of effective HCC chemoprevention and treatment.

Karyotype of cryopreserved bone marrow cells

The analysis of chromosomal abnormalities is important for the study of hematological neoplastic disorders since it facilitates classification of the disease. The ability to perform chromosome analysis of cryopreserved malignant marrow or peripheral blast cells is important for retrospective studies. In the present study, we compared the karyotype of fresh bone marrow cells (20 metaphases) to that of cells stored with a simplified cryopreservation method, evaluated the effect of the use of granulocyte-macrophage colony-stimulating factor (GM-CSF) as an in vitro mitotic index stimulator, and compared the cell viability and chromosome morphology of fresh and cryopreserved cells whenever possible (sufficient metaphases for analysis). Twenty-five bone marrow samples from 24 patients with hematological disorders such as acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, chronic myeloid leukemia, megaloblastic anemia and lymphoma (8, 3, 3, 8, 1, and 1 patients, respectively) were selected at diagnosis, at relapse or during routine follow-up and one sample was obtained from a bone marrow donor after informed consent. Average cell viability before and after freezing was 98.8 and 78.5 percent, respectively (P < 0.05). Cytogenetic analysis was successful in 76 percent of fresh cell cultures, as opposed to 52 percent of cryopreserved samples (P < 0.05). GM-CSF had no proliferative effect before or after freezing. The morphological aspects of the chromosomes in fresh and cryopreserved cells were subjectively the same. The present study shows that cytogenetic analysis of cryopreserved bone marrow cells can be a reliable alternative when fresh cell analysis cannot be done, notwithstanding the reduced viability and lower percent of successful analysis that are associated with freezing