AdhMMP8 Vector Administration in Muscle: An Alternate Strategy to Regress Hepatic Fibrosis.

The development of several vaccines against the SARS-CoV2 virus and their application in millions of people have shown efficacy and safety in the transfer of genes to muscle turning this tissue into a protein-producing factory. Established advanced liver fibrosis, is characterized by replacement of hepatic parenchyma by tissue scar, mostly collagen type I, with increased profibrogenic and proinflammatory molecules gene expression. Matrix metalloproteinase 8 (MMP-8) is an interstitial collagen-degrading proenzyme acting preferentially on collagen type I when activated. This study was carried out to elucidate the effect of an intramuscularly delivered adenoviral vector containing proMMP-8 gene cDNA (AdhMMP8) in male Wistar rats with experimental advanced liver fibrosis induced by thioacetamide. Therapeutic effects were monitored after 1, 2, or 3 weeks of a single dose (3 × 1011 vp/kg) of AdhMMP8. Circulating and liver concentration of MMP-8 protein remained constant; hepatic fibrosis decreased up to 48%; proinflammatory and profibrogenic genes expression diminished: TNF-α 2.28-fold, IL-1 1.95-fold, Col 1A1 4-fold, TGF-ß1 3-fold and CTGF 2-fold; and antifibrogenic genes expression raised, MMP-9 2.8-fold and MMP-1 10-fold. Our data proposes that the administration of AdhMMP8 in muscle is safe and effective in achieving liver fibrosis regression at a comparable extent as when the adenoviral vector is delivered systemically to reach the liver, using a minimally invasive procedure.

Nrf2 and Snail-1 in the prevention of experimental liver fibrosis by caffeine.

AIM: To determine the molecular mechanisms involved in experimental hepatic fibrosis prevention by caffeine (CFA). METHODS: Liver fibrosis was induced in Wistar rats by intraperitoneal thioacetamide or bile duct ligation and they were concomitantly treated with CFA (15 mg/kg per day). Fibrosis and inflammatory cell infiltrate were evaluated and classified by Knodell index. Inflammatory infiltrate was quantified by immunohistochemistry (anti-CD11b). Gene expression was analyzed by quantitative reverse transcription-polymerase chain reaction for collagen I (Col-1), connective tissue growth factor (CTGF), transforming growth factor ß1 (TGF-ß1), tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), IL-6, superoxide dismutase (SOD) and catalase (CAT). Activation of Nrf2 and Snail-1 was analyzed by Western-blot. TNF-α expression was proved by enzyme-linked immunosorbant assay, CAT activity was performed by zymography. RESULTS: CFA treatment diminished fibrosis index in treated animals. The Knodell index showed both lower fibrosis and necroinflammation. Expression of profibrogenic genes CTGF, Col-1 and TGF-ß1 and proinflammatory genes TNF-α, IL-6 and IL-1 was substantially diminished with CFA treatment with less CD11b positive areas. Significantly lower values of transcriptional factor Snail-1 were detected in CFA treated rats compared with cirrhotic rats without treatment; in contrast Nrf2 was increased in the presence of CFA. Expression of SOD and CAT was greater in animals treated with CFA showing a strong correlation between mRNA expression and enzyme activity. CONCLUSION: Our results suggest that CFA inhibits the transcriptional factor Snail-1, down-regulating profibrogenic genes, and activates Nrf2 inducing antioxidant enzymes system, preventing inflammation and fibrosis.

Epigenetic therapy with hydralazine and magnesium valproate reverses imatinib resistance in patients with chronic myeloid leukemia.

UNLABELLED: The epigenetic drugs hydralazine and valproate were administered in a compassionate manner to 8 patients with chronic myeloid leukemia (CML) refractory to imatinib. Two patients had a complete hematologic response (25%),1 major cytogenetic response, 1 complete cytogenetic response (25% any cytogenetic response), and 3 (37.5%)stable disease. No grade 3 or 4 toxicity was observed. These results show the ability of epigenetic therapy to revert imatinib resistance. BACKGROUND: Epigenetic alterations participate in the development of acquired resistance to imatinib, hence, the DNA methylation, and histone deacetylase inhibitors hydralazine and valproate, respectively, has the potential to overcome it. PATIENT AND METHODS: A series of 8 patients with chronic myeloid leukemia (CML) refractory to imatinib mesylate with no access to second-generation tyrosine kinase inhibitors were treated with hydralazine and valproate in a compassionate manner. Clinical efficacy and safety of these drugs added to imatinib mesylate were evaluated. RESULTS: Two patients were in the blast phase, 5 were in the accelerated phase, and 1 was in the chronic phase. All the patients continued with the same dose of imatinib that they had been receiving at the time of development of resistance, with a median dose of 600 mg daily (range, 400-800 mg). The median time from diagnosis of CML to the start of hydralazine and valproate was 53.6 months (range, 19-84 months). Two (25%) patients had a complete hematologic response, one (12.5%) had an major cytogenetic response, and one (12.5%) had a complete cytogenetic response. Three (37.5%) patients had stable disease, and only one (12.5%) patient failed to respond. At a median follow-up time of 18 months (range, 3-18 months), the median survival had not been reached, and the projected overall survival was 63%. All the patients had mild neurologic toxicity, including distal tremor and somnolence. No grade 3 or 4 toxicity was observed. CONCLUSIONS: Our results suggest that the epigenetic drugs hydralazine and valproate revert the resistance to imatinib in patients with CML.

Genetic polymorphisms of genes coding to alcohol-metabolizing enzymes in western Mexicans: association of CYP2E1*c2/CYP2E1*5B allele with cirrhosis and liver function.

BACKGROUND: Alcoholic cirrhosis constitutes a major public health problem in the world where ADH1B, ALDH2, and CYP2E1 polymorphisms could be playing an important role. We determined ADH1B*2, ALDH2*2, and CYP2E1*c2 allele frequencies in healthy control individuals (C) and patients with alcoholic cirrhosis (AC) from western Mexico. METHODS: Ninety C and 41 patients with AC were studied. Genotype and allele frequency were determined through polymerase chain reaction-restriction fragment length polymorphisms. RESULTS: Polymorphic allele distribution in AC was 1.6%ADH1B*2, 0.0%ALDH2*2, and 19.5%CYP2E1*c2; in C: 6.1%ADH1B*2, 0%ALDH2*2, and 10.6%CYP2E1*c2. CYP2E1*c2 polymorphic allele and c1/c2 genotype frequency were significantly higher (p < 0.05 and p < 0.01, respectively) in patients with AC when compared to C. Patients with AC, carrying the CYP2E1*c2 allele, exhibited more decompensated liver functioning evaluated by total bilirubin and prothrombin time, than c1 allele carrying patients (p < 0.05). Cirrhosis severity, assessed by Child's Pugh score and mortality, was higher in patients carrying the c2 allele, although not statistically significant. CONCLUSIONS: In this study, CYP2E1*c2 allele was associated with susceptibility to AC; meanwhile, ADH1B*2 and ALDH2*2 alleles were not. CYP2E1*c2 allele was associated with AC severity, which could probably be attributed to the oxidative stress promoted by this polymorphic form. Further studies to clearly establish CYP2E1*c2 clinical relevance in the development of alcohol-induced liver damage and its usefulness as a probable prognostic marker, should be performed. Also, increasing the number of patients and including a control group conformed by alcoholic patients free of liver damage may render more conclusive results. These findings contribute to the understanding of the influence of gene variations in AC development among populations, alcohol metabolism, and pharmacogenetics.

Polymorphisms of alcohol metabolizing enzymes in indigenous Mexican population: unusual high frequency of CYP2E1*c2 allele.

BACKGROUND: Alcohol abuse represents the major identified etiological factor of cirrhosis in México. ADH1B, ALDH2, and CYP2E1 have been considered candidate genes in alcohol-related diseases. Controversial results probably due to ethnic differences, among other factors, have been reported. Mexican Mestizos (MES) derive from the combination of indigenous, Spaniard, and African genes. Huichols (HUI) constitute an indigenous group from western Mexico with no racial admixture. We determined ADH1B*2, ALDH2*2, and CYP2E1*c2 allele frequencies in healthy HUI and MES from western Mexico. Lipid and hepatic profile were also carried out. METHODS: One hundred and one HUI and 331 MES subjects were studied. Genotype and allele frequency were assessed through polymerase chain reaction-restriction fragment length polymorphism after DNA isolation from peripheral leukocytes. Commercial kits for lipid and hepatic determinations were used. RESULTS: Polymorphic allele distribution in HUI was: 0%ADH1B*2, 0.5%ALDH2*2, 51.5%CYP2E1*c2; in MES: 3.4%ADH1B*2, 0%ALDH2*2, 16.1%CYP2E1*c2. Frequency of ADH1B*2 was statistically (p < 0.001) lower in HUI than MES. CYP2E1*c2 polymorphic allele was significantly higher (p < 0.0001) in HUI than MES. Hepatic profile was normal in both groups. HUI showed a better lipid profile than MES independently of genotype. CONCLUSIONS: Huichols exhibited the highest CYP2E1*c2 allele frequency of the world documented up to this date; meanwhile, ADH1B*2 and ALDH2*2 were practically absent. This feature could be useful in the understanding of Mexican population gene composition, alcohol metabolism, and alcoholic liver disease development. However, further association studies are necessary. The heterogeneity of Mexican population was evidenced by the significantly different distribution of CYP2E1*c2 allele observed among different regions of the country. Lipid and hepatic values were not associated to genotype. This report constitutes the first study dealing with gene polymorphisms of alcohol metabolizing enzymes conducted in HUI.