Losartan reduces trinitrobenzene sulphonic acid-induced colorectal fibrosis in rats.

BACKGROUND: Intestinal fibrosis is a challenging clinical condition in several fibrostenosing enteropathies, particularly Crohn's disease. Currently, no effective preventive measures or medical therapies are available for intestinal fibrosis. Fibrosis, due to an abnormal accumulation of extracellular matrix proteins, is a chronic and progressive process mediated by cell/matrix/cytokine and growth factor interactions, but may be a reversible phenomenon. Of the several molecules regulating fibrogenesis, transforming growth factor-beta 1 (TGF-b1) appears to play a pivotal role; it is strongly induced by the local activation of angiotensin II. The levels of both TGF-b1 and angiotensin II are elevated in fibrostenosing Crohn's disease. AIMS: To evaluate the in vivo effect of losartan - an angiotensin II receptor antagonist - on the course of chronic colitis-associated fibrosis and on TGF-b1 expression. METHODS: Colitis was induced by intrarectal instillation of trinitrobenzene sulphonic acid (TNBS) (15 mg/mL) while losartan was administered orally daily by gavage (7 mg/kg/day) for 21 days. Three groups of rats were evaluated: control (n=10); TNBS treated (n=10); and TNBS + losartan treated (n=10). Inflammation and fibrosis of the colon were evaluated by macro- and microscopic score analysis. Colonic TGF-b1 levels was measured using ELISA. RESULTS: Twenty-one days after induction, losartan significantly improved the macro- and microscopic scores of fibrosis in the colonic wall and reduced TGF-b1 concentration. CONCLUSIONS: Prophylactic oral administration of losartan reduces the colorectal fibrosis complicating the TNBS-induced chronic colitis, an effect that appears to be mediated by a downregulation of TGF-b1 expression.

Femtosecond laser: a new intradermal DNA delivery method for efficient, long-term gene expression and genetic immunization.

A femtosecond laser beam gene transduction (SG-LBGT) system is described as a novel and efficient method of intradermal (i.d.) nonviral gene delivery in mice by permeabilizing cells utilizing femtosecond laser pulses. Using this approach, significant gene expression and efficient dermal transduction lasting for >7 months were obtained. The ability of this new DNA gene transfer method to enhance genetic vaccination was tested in BALB/C mice. A single i.d. injection of a plasmid (10 microg) containing the hepatitis B virus (HBV) surface antigen (HBsAg), followed by pulses of laser, induced high titers of HBsAg-specific antibodies lasting for >210 days and increased levels of IgG1, IgG2a, IFNgamma, and IL-4, indicating the activation of both Th1 and Th2 cells. Moreover, mice vaccinated using the SG-LBGT followed by challenge with pHBV showed increased protection against viral challenge, as detected by decreased levels of HBV DNA, suggesting an efficient Th1 effect against HBV-infected replicating cells. Tumor growth retardation was induced in vaccinated mice challenged with an HBsAg-expressing syngeneic tumor. In most of the parameters tested, administration of plasmid followed by laser application was significantly more effective and prolonged than that of plasmid alone. Tissue damage was not detected and integration of the plasmid into the host genomic DNA probably did not occur. We suggest that the LBGT method is an efficient and safe technology for in vivo gene expression and vaccination and emphasizes its potential therapeutic applications for i.d. nonviral gene delivery.

Treatment of chronic hepatitis B virus infection via oral immune regulation toward hepatitis B virus proteins.

OBJECTIVES: Hepatitis B virus (HBV) is a noncytopathic virus, and hepatocellular injury is mediated by a defective host antiviral immune response. We have previously shown that antiviral immunity can be modulated through oral feeding of viral proteins. The aims of this study were to determine the safety and efficacy of treatment of patients with chronic HBV by means of p.o. administration of HBV envelope proteins. METHODS: A total of 42 chronic HBV patients were treated p.o. with HBV envelope proteins (HBsAg+preS1+preS2), three times/wk for 20-30 wk, and followed for an additional 20 wk. Patients were monitored for HBV-DNA levels, liver enzymes, and liver histology. HBV-directed T cell immune modulation was assessed in vitro by HBV specific T cell-proliferation, cytotoxicity, IFN gamma, and IL10 ELISPOT assays, and reverse transcription-polymerase chain reaction cytokines assay. RESULTS: Favorable response in one of the primary endpoints was achieved in 28/42 patients (66.6%) by means of p.o. immune regulation. A significant decrease in viral load was observed in 15 patients (35.7%). HBsAg/HBcAg biopsy scores improved in 41% and 57.1% of patients, respectively. Histological improvement in liver necroinflammatory score was noted in 12/40 patients (30%). In all, 80% showed biochemical response. Five of 19 HBeAg positive patients (26.3%) became negative for HBeAg. A favorable augmentation in anti-HBV specific T cell response, with increased HbsAg specific T cell proliferation (78%), cytotoxicity (75%), and IFN gamma positive T cell clones (62.9%) was noted. In addition, a decrease in the IL10 gamma positive T cell clones was achieved (48.1%). Natural killer T (NKT) lymphocytes increased significantly in all treated patients. CONCLUSIONS: Immune regulation of the anti-HBV immune response via p.o. administration of HBV envelope proteins alleviated the immune-mediated liver injury while augmenting the effective antiviral immunity.

Cytokine gene polymorphisms in patients infected with hepatitis B virus.

OBJECTIVE: Cytokines play a key role in the regulation of the immune response. The maximal capacity of cytokine production varies among individuals and correlates with the polymorphism in the cytokine gene promoters. The aim of this study was to characterize gene polymorphism in patients with chronic hepatitis B virus (HBV) infection and to determine the different patterns in patient subgroups. METHODS: The study population consisted of 77 patients with chronic HBV infection (23 low-level HBV replicative carriers, 23 compensated high-level HBV replicative carriers, 21 decompensated liver transplant candidates, and 10 patients with documented hepatocellular carcinoma). The genetic profile of five cytokines was analyzed by polymerase chain reaction-sequence-specific primer (SSP), and subjects were genotyped as high or low producers of tumor necrosis factor-alpha and interleukin (IL)-6, and as high, intermediate, or low producers of transforming growth factor-beta(1), interferon (IFN)-gamma, and IL-10 based on single nucleotide substitutions. The control group included 10 healthy individuals who recovered from HBV infection and 48 healthy controls. RESULTS: A highly statistically significant difference in the distribution of the IFN-gamma gene polymorphism (at position +879) was observed between patients with chronic HBV infection and controls. The majority of the patients (65.2%) exhibited the potential to produce low levels of IFN-gamma (A/A genotype) compared with 37.5% of the control group (p = 0.003). Healthy individuals who recovered from HBV infection had a similar distribution of IFN-gamma gene polymorphism as the healthy controls. No statistically significant difference in IFN-gamma production was found between patients with low- and high-level HBV replication and between compensated and decompensated patients. There was also no statistically significant difference in the genetic ability to produce tumor necrosis factor-alpha (at position -308), IL-6 (at position -174), IL-10 (at position -1082, -819, and -592), and transforming growth factor-beta(1) (at position +10 and +25). CONCLUSION: These findings suggest an association between the genetic ability to produce low levels of IFN-gamma and the susceptibility to develop chronic HBV infection.

Genotypic and phenotypic resistance: longitudinal and sequential analysis of hepatitis B virus polymerase mutations in patients with lamivudine resistance after liver transplantation.

OBJECTIVE: Lamivudine-resistant strains appear in 27-62.5% of liver transplant recipients treated with lamivudine for hepatitis B virus (HBV) recurrence, and may lead to failure of antiviral therapy. In an extension of our previous study, we investigated the molecular events associated with the emergence of lamivudine-resistant mutants in this population. METHODS: Sequential serum samples from 10 consecutive patients with lamivudine resistance after liver transplantation were analyzed for viral genotype, precore mutants, and viral polymerase gene mutants (L528M, M552V, M552I) using restriction fragment length polymorphism. Quantitative analysis of HBV DNA was performed using hybridization assay and polymerase chain reaction. RESULTS: Eight patients (80%) were infected with genotype D and two (20%) with genotype C. Polymerase mutants (genotypic resistance) were identified in all the patients. Phenotypic resistance (rise in serum HBV DNA titers above the detection limit of the hybridization assay) developed in five patients (50%); of the remainder, three (30%) did not have phenotypic resistance, and two were primary nonresponders. Genotypic resistance was detected earlier than phenotypic resistance (median 285 days [range 42-510] vs median 387 days [range 320-420], p = 0.055). In five patients (50%), the emergence of the YMDD mutants took over the wild type; in three (30%), the YMDD mutant took over the wild type, but the wild type re-emerged during lamivudine therapy; and in two (20%), the YMDD mutants were detected in a mixture with the wild type (in different percentages). The mean pretreatment serum ALT level was significantly lower in the patients who did not develop phenotypic resistance (p = 0.0002). The M552I pure viral population was found mainly in these patients, and all retained stable graft function (median follow-up 33 months). A high pretreatment HBV DNA level (>50 x 10(6) copies/ml) was highly statistically significantly correlated with the rapid occurrence of phenotypic resistance (r = -0.90, p = 0.04). CONCLUSIONS: We reached the following conclusions: 1) In our area, liver transplant recipients who develop resistance to lamivudine given for recurrent HBV infection seem to be mainly infected with genotype D. 2) Re-emergence of the wild type can occur during lamivudine therapy. 3) Genotypic resistance precedes phenotypic resistance, although phenotypic resistance does not always follow genotypic resistance. 4) Quantitative determination of viremia and analysis of polymerase gene mutants are recommended for monitoring antiviral therapy of liver transplant patients with HBV reinfection in the graft.

Role of anti-tumor necrosis factor-alpha in ischemia/reperfusion injury in isolated rat liver in a blood-free environment.

BACKGROUND: Warm ischemia/reperfusion injury during liver transplantation is the most important cause of primary nonfunction of liver allografts. Tumor-necrosis factor (TNF)-alpha apparently mediates tissue damage by inducing apoptosis and/or necrosis in liver transplants. The aim of the study was to determine, using an isolated rat liver model, if pretreatment with anti-TNF-alpha monoclonal antibodies can attenuate ischemia/reperfusion liver injury. Specifically, its effect on liver cell apoptosis through the modulation of caspase activity was examined in a blood-free environment. METHODS: Isolated rat livers were perfused with Krebs-Henseleit solution and randomly divided into three groups: (1) continuous perfusion for 165 min (control); (2) perfusion for 90 min, break for 60 min (ischemia), and reperfusion for 15 min; (3) as with group 2, but with administration of monoclonal mouse anti-rat TNF-alpha monoclonal antibodies before induction of ischemia. Caspase-3- and -9-like activity was measured by fluorometric assay, and apoptotic cells were identified by morphological criteria and application of the terminal deoxnucleotidyl transferase-mediated dUTP nick-end-labeling (Tunel) assay. RESULTS: Portal pressure increased significantly in group 2 (14.8+/-2.3 mm Hg) compared to group 3, which showed no change (P<0.05). Significant amounts of TNF-alpha were detected in the effluent in group 2 at 1 min of reperfusion (147+/-8.9 pg/ml) compared to group 3 (30+/-6.7 pg/ml, P<0.05). Statistically significant reductions in liver enzyme levels were also noted in the animals pretreated with TNF-alpha antibodies (P<0.02). Caspase-3 and -9 activity was significantly decreased (270 and 160%, respectively) in group 3 compared to group 2 (P<0.005 and <0.05, respectively). A significant reduction in postischemic hepatic injury was noted on Tunel assay: many apoptotic hepatocyte cells were detected in group 2 but not in livers pretreated with monoclonal mouse anti-TNF-alpha antibodies (group 3). CONCLUSIONS: Neutralization with specific monoclonal antibodies against TNF before ischemia induction can attenuate postischemic hepatic injury. Apoptotic injury seems to be ameliorated through modulation of caspase-3- and -9-like activity.

A mutation in the peroxisome proliferator-activated receptor gamma-binding site in the gene for the cytosolic form of phosphoenolpyruvate carboxykinase reduces adipose tissue size and fat content in mice.

Regulation of the turnover of triglycerides in adipose tissue requires the continuous provision of 3-glycerophosphate, which may be supplied by the metabolism of glucose or by glyceroneogenesis, the de novo synthesis of 3-glycerophosphate from sources other than hexoses or glycerol. The importance of glyceroneogenesis in adipose tissue was assessed in mice by specifically eliminating the expression of the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C), an enzyme that plays a pivotal role in the pathway. To accomplish this, we mutated the binding site for the peroxisome proliferator-activated receptor gamma (PPAR gamma) called the peroxisome proliferator-activated receptor element (PPARE), in the 5' flanking region of the PEPCK-C gene in the mouse by homologous recombination. The mutation abolished expression of the gene in white adipose tissue and considerably reduced its expression in brown adipose tissue, whereas the level of PEPCK-C mRNA in liver and kidney remained normal. Epididymal white adipose tissue from these mice had a reduced triglyceride deposition, with 25% of the animals displaying lipodystrophy. There was also a greatly reduced level of lipid accumulation in brown adipose tissue. A strong correlation between the hepatic content of triglycerides and the size of the epididymal fat pad in PPARE(-/-) mice suggests that hepatic triglyceride synthesis predominantly utilizes free fatty acids derived from the adipose tissue. Unlike other models, PPARE(-/-) mice with lipodystrophy did not exhibit the lipodystrophy-associated features of diabetes and displayed only moderate hyperglycemia. These studies establish the importance of the PPARE site for PEPCK-C gene expression in adipose tissue and the role of PEPCK-C in the regulation of glyceroneogenesis, a pathway critical for maintaining the deposition of triglycerides in adipose tissue.