Antitumoral gene-based strategy involving nitric oxide synthase type III overexpression in hepatocellular carcinoma.

Hepatocellular carcinoma develops in cirrhotic liver. The nitric oxide (NO) synthase type III (NOS-3) overexpression induces cell death in hepatoblastoma cells. The study developed gene therapy designed to specifically overexpress NOS-3 in cultured hepatoma cells, and in tumors derived from orthotopically implanted tumor cells in fibrotic livers. Liver fibrosis was induced by CCl4 administration in mice. The first-generation adenoviruses were designed to overexpress NOS-3 or green fluorescent protein, and luciferase complementary DNA under the regulation of murine alpha-fetoprotein (AFP) and Rous Sarcoma Virus (RSV) promoters, respectively. Both adenovirus and Hepa 1-6 cells were used for in vitro and in vivo experiments. Adenoviruses were administered through the tail vein 2 weeks after orthotopic tumor cell implantation. AFP-NOS-3/RSV-luciferase increased oxidative-related DNA damage, p53, CD95/CD95L expression and caspase-8, -9 and -3 activities in cultured Hepa 1-6 cells. The increased expression of CD95/CD95L and caspase-8 activity was abolished by Nω-nitro-l-arginine methyl ester hydrochloride, p53 and CD95 small interfering RNA. AFP-NOS-3/RSV-luciferase adenovirus increased cell death markers, and reduced cell proliferation of established tumors in fibrotic livers. The increase of oxidative/nitrosative stress induced by NOS-3 overexpression induced DNA damage, p53, CD95/CD95L expression and cell death in hepatocellular carcinoma cells. The effectiveness of the gene therapy has been demonstrated in vitro and in vivo.

An Experimental Study on the Use of Calcium Alginate to Heal Colonic Anastomoses.

BACKGROUND: Anastomotic leak is considered the major complication following abdominal surgery. In recent years, the use of a variety of sealing materials for the prevention of leaks has been analyzed. Different biomaterials have been employed as scaffolds to favour tissue repair and regeneration. Among these materials we must mention alginate, a natural polymer with different applications as temporary supporting matrix. The aim of the present study is to evaluate the behavior of both alginate-impregnated sutures and lyophilized alginate sponges in the healing process of colonic anastomes using an experimental animal model. MATERIAL AND METHODS: A preliminary study was undertaken to select the adequate scaffold. Animals (n = 45) were distributed into three groups: control (colonic anastomosis using non-continuous 5-0 Polyglactin 910 suture), suture (colonic anastomosis using suture impregnated with alginate gel at 4%) and sponge (colonic anastomosis using suture reinforced with lyophilized alginate sponge). The macroscopic and histological variables were assessed at 4, 8 and 12 days after surgical intervention. RESULTS: No statistically significant differences have been observed between the groups during the analysis of macroscopic variables. Animals with sponge implantation showed a greater degree of epithelial reepithalization, less acute and chronic inflammation and greater collagen deposit. CONCLUSIONS: The use of lyophilized alginate sponges to reinforce colonic anastomoses in an animal model reduces inflammation and promotes the earlier formation of greater collagen deposits without increasing the number of adhesions or the incidence of stenosis.

Regulation of cell death receptor S-nitrosylation and apoptotic signaling by Sorafenib in hepatoblastoma cells.

Nitric oxide (NO) plays a relevant role during cell death regulation in tumor cells. The overexpression of nitric oxide synthase type III (NOS-3) induces oxidative and nitrosative stress, p53 and cell death receptor expression and apoptosis in hepatoblastoma cells. S-nitrosylation of cell death receptor modulates apoptosis. Sorafenib is the unique recommended molecular-targeted drug for the treatment of patients with advanced hepatocellular carcinoma. The present study was addressed to elucidate the potential role of NO during Sorafenib-induced cell death in HepG2 cells. We determined the intra- and extracellular NO concentration, cell death receptor expression and their S-nitrosylation modifications, and apoptotic signaling in Sorafenib-treated HepG2 cells. The effect of NO donors on above parameters has also been determined. Sorafenib induced apoptosis in HepG2 cells. However, low concentration of the drug (10nM) increased cell death receptor expression, as well as caspase-8 and -9 activation, but without activation of downstream apoptotic markers. In contrast, Sorafenib (10 µM) reduced upstream apoptotic parameters but increased caspase-3 activation and DNA fragmentation in HepG2 cells. The shift of cell death signaling pathway was associated with a reduction of S-nitrosylation of cell death receptors in Sorafenib-treated cells. The administration of NO donors increased S-nitrosylation of cell death receptors and overall induction of cell death markers in control and Sorafenib-treated cells. In conclusion, Sorafenib induced alteration of cell death receptor S-nitrosylation status which may have a relevant repercussion on cell death signaling in hepatoblastoma cells.

Antitumor efficacy of mammalian target of rapamycin inhibitor therapy in liver transplant recipients with oncological disease: a case-control study.

INTRODUCTION: The reported incidences of de novo malignancy following orthotopic liver transplantation (OLT) are significantly greater than those in the general population. We have analyzed the efficacy of mammalian target of rapamycin inhibitor (mTORi) as immunosuppressant therapy in patients with de novo malignancies or those engrafted because of a primary liver cancer. METHODS: We performed a case-control study of patients with hepatocellular carcinoma (HCC; n = 119), cholangiocarcinoma (n = 1) or de novo malignancies (n = 73). Thirty-seven patients with these tumors were treated with mTORi, and 167, with calcineurin inhibitors (CNI). Switching to mTORi was performed progressively, withdrawing the CNI over 15 days, until obtaining levels of 5-10 ng/dL. RESULTS: No incidence of rejection, serious adverse events, or death was observed with an overall actuarial survival of 68.5% in the mTORi group versus 45.7% among the CNI group. Overall rates of tumor recurrence were 15.2% and 36.8%, respectively (P < .05). Among patients with HCC, survival was 100% of mTORi with and 61.5% among CNI patients, with tumor recurrence rates of 6.2% and 19.1%, respectively (P < .05). DISCUSSION: Surprising differences in survival and tumor recurrence rates were observed among the mTORi-treated group compared with controls. Switching from CNI to mTORi immunosuppressant therapy appeared to be safe. It seems to be reasonable to employ this strategy in liver transplant patients with primary hepatic or "de novo" neoplasms.

Results of liver transplantation with donors older than 70 years: a case-control study.

Older donors are a growing part of the total pool but no definite consensus exists on the age limit for their acceptance. This retrospective case-control unicenter study compared the outcomes of 72 orthotopic liver transplantations (OLTs) from April 1990 to April 2010 using donors older than 70 years versus 738 chronologically correlated OLTs performed with donors younger than 60 years. The percentage of refusal was greater among older than younger donors (48.2 vs 14.3%; P < .001). No difference was observed in mean cold ischemia times between older (370.5 minutes) versus younger groups (389.2 minutes). or in postoperative complications of rejection or renal insufficiency except for sepsis and mortality. Long-term survival was lower among transplant recipients from donors older than 70 years (P = .001) and these cases showed more blood requirements associated with prolonged cold ischemia (P = .02). Multivariate analysis revealed graft dysfunction, mortality, and reduced survival to be associated with donor weight and recipient MELD (Model for End-stage Liver Disease) (P < .05). Interestingly, the mortality related to hepatitis C virus recurrence was not greater among patients whose donors were older than 70. Septuagenarians' livers can be used safely, but careful donor and recipient evaluation are required to avoid additional risk factors.

Liver transplantation in patients with cryptogenic cirrhosis: long-term follow-up.

OBJECTIVE: The objective of this study was to evaluate long-term survival, histological diagnoses, and mobility of patients with cryptogenic cirrhosis (CC) treated with orthotopic liver transplantation (OLT). PATIENTS AND METHODS: We performed a retrospective analysis of 35 patients who underwent transplantation with CC among 800 OLT patients. There were no differences in gender, mean age of 47 years, average MELD (Model for End-stage Liver Disease) of 16, and hepatocellular carcinoma incidence (8%). RESULTS: In 28.6% of patients, the diagnosis of CC was wrong. There was no incidence of an acute rejection episode and a low incidence of complications, although the postoperative mortality rate was 20%, of chronic rejection was 25%, and recurrence of disease was 4%. Cumulative at 3-, 5-, and 10-year survivals were lower than the other OLT. Survival was lower in patients receiving suboptimal grafts. CONCLUSIONS: One of 3 patients who underwent transplantation for CC had a specific etiologic diagnosis. The chronic rejection rate and postoperative mortality rate were higher than other etiologies, and survivals at 5, 10, and 15 years were lower than other OLT.

Effect of melatonin on myocardial oxidative stress induced by experimental obstructive jaundice.

OBJECTIVE: melatonin has been demonstrated to have active antioxidant properties in different tissues during experimental cholestasis. The aim of this research was to study myocardial oxidative stress on obstructive jaundice, and to analyze the effect of melatonin on myocardial oxidative lesions. MATERIAL AND METHODS: we achieved cholestasis by ligature and sectioning of the main bile duct. Melatonin was administered intraperitoneally (500 microg/kg/day). We measured malondialdehyde (MDA), reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione peroxydase (GPx) antioxidant enzyme levels in the heart tissue. RESULTS: obstructive cholestasis increased MDA and decreased GSH as well as all antioxidant enzymes. Melatonin administration significantly decreased MDA values, and increased GSH and antioxidant enzymes on the icteric animal myocardium. CONCLUSIONS: melatonin treatment prevents oxidative stress in the cardiac tissue as induced by experimental cholestasis.

Alteration of the renal regulatory hormonal pattern during experimental obstructive jaundice.

OBJECTIVE: The alteration of hormones regulating sodium and water status is related to renal failure in obstructive jaundice (OJ). EXPERIMENTAL DESIGN: OJ was induced by common bile duct ligation. Samples were obtained from the control (SO) and OJ groups at 24 and 72 hours, and at 7 days. Different parameters related to biliary obstruction, liver and renal injury, and vasoactive mediators such as renin, aldosterone, endothelin-1 (ET-1) and prostaglandin E2 (PGE2) were studied. RESULTS: Bile duct ligation caused an increase in total bilirubin (p < 0.001) and alkaline phosphatase (AP) (p < 0.001). The SO and OJ groups had the same values for diuresis, renin, and creatinine clearance at 24 h. However, animals with OJ had a lower sodium concentration in urine than SO animals (p < 0.01), as well as an increase in aldosterone levels (p < 0.03). ANP levels were moderately increased during OJ but did not reach statistical significance when compared to the SO group. In contrast, OJ animals showed a rise in serum ET-1 concentration (p < 0.001) and increased PGE2 in urine (p < 0.001). CONCLUSIONS: Biliary obstruction induced an increase in ET-1 release and PGE2 urine excretion. These hormones might play a role during the renal complications associated with renal disturbances that occur during OJ.

Anti-TNF-alpha treatment and bile duct drainage restore cellular immunity and prevent tissue injury in experimental obstructive jaundice.

Several experimental studies of obstructive jaundice (OJ) have shown the presence of immunosuppressive state associated with the rise of tumor necrosis factor-alpha (TNF-alpha) concentration in plasma. The present study evaluates the impact of anti-TNF- alpha administration or bile duct drainage on the inflammatory response, liver injury and renal insufficiency in obstructed rats. OJ was induced by the ligation of bile duct in Wistar rats. The parameters were determined at 14 and 21 days after OJ. Two additional groups of animals were treated with anti-TNF-alpha antibodies or submitted to bile duct drainage at 14 days, and sacrificed 21 days after OJ. Cholestasis decreased glucose, and enhanced urea, creatinin, bilirubin and transaminases. Cholestasis increased the number of different inflammatory cells (T and B lymphocytes, and monocytes-macrophages) but reduced the expression of the corresponding cellular activation markers. This low responsiveness of the inflammatory cells was related to a decreased free radical production and phagocytic activity of cells. Anti-TNF-alpha and bile duct drainage reduced tissue injury, and prevented the reduction of the number and activity of T lymphocytes and phagocytic cells observed at the advanced stages of cholestasis. In conclusion, anti-TNF- alpha and bile duct drainage improved cell immunodeficiency, and reduced liver injury, cholestasis and renal insufficiency in experimental OJ.

Multivariate analysis of factors associated with renal dysfunction in patients with obstructive jaundice.

BACKGROUND: The aim was to evaluate the factors determining preoperative renal dysfunction in patients with obstructive jaundice. METHODS: In a prospective cross-sectional observational study, 63 patients, 27 with benign and 36 with malignant obstructive jaundice, were investigated at admission and compared with 25 healthy control subjects. Variables analysed included extracellular body water (ECW) compartment, plasma levels of aldosterone, renin, atrial natriuretic peptide, vasopressin, nitric oxide, endothelin (ET) 1 and prostaglandin E2 (PGE2), urinary nitric oxide and PGE2, serum albumin and renal function. RESULTS: The metabolic profile of obstructive jaundice was characterized by a depletion of the ECW (P = 0.004), and increased plasma levels of atrial natriuretic peptide (P < 0.001), ET-1 (P = 0.044), vasopressin (P = 0.017), aldosterone (P = 0.005) and renin (P = 0.001). Increased plasma (P < 0.001) and urinary (P = 0.001) PGE2 levels were also found. Fifty-four per cent of patients had a creatinine clearance of less than 70 ml/min. In multivariate analysis, serum bilirubin, renin, ET-1, PGE2, decreased urinary sodium excretion and age were identified as predictors of renal dysfunction. CONCLUSIONS: Renal dysfunction in patients with obstructive jaundice was associated with the degree of biliary obstruction, age of the patient and reduced urinary sodium excretion. These alterations were closely related to derangements in sodium- and water-regulating hormones.

Randomized clinical trial of the effect of intravenous fluid administration on hormonal and renal dysfunction in patients with obstructive jaundice undergoing endoscopic drainage.

BACKGROUND: Renal dysfunction in patients with biliary obstruction is associated with extracellular water depletion. This study examined the effect of preoperative saline infusion before biliary drainage on hormonal and renal functional derangements in patients with obstructive jaundice. METHODS: In a randomized study, 49 patients with malignant obstructive jaundice were investigated at baseline, on the day of drainage, and at 24 h, 72 h and 7 days after internal endoscopic biliary drainage. Patients were randomized to receive (n = 22) or not to receive (n = 27) 3000 ml normal saline intravenously for 24 h before drainage. Variables analysed included extracellular water volume, creatinine clearance, and serum levels of aldosterone, renin, atrial natriuretic peptide (ANP), vasopressin and albumin. RESULTS: Preoperative saline infusion produced a rise in creatinine clearance, diuresis, ANP concentration and extracellular water volume but this did not translate into better recovery of renal function after operation. Drainage produced a fall in creatinine clearance in all patients, but hormonal and renal function had recovered by 2 days after restoration of bile flow, independently of preoperative hydration. CONCLUSION: Fluid administration expands the extracellular water compartment before drainage but fails to improve renal function after drainage. Definitive improvement in endocrine and renal function requires the restoration of bile flow into the duodenum.

Melatonin versus vitamin E as protective treatment against oxidative stress after extra-hepatic bile duct ligation in rats.

The aims of the present study were first to compare the effects of melatonin and vitamin E on the cholestasis syndrome and their protective effect on liver injury, and second, to evaluate the activity of antioxidant enzymes after treatment with these antioxidant drugs. Cholestasis was achieved in adult male Wistar rats by double ligature and section of the extra-hepatic biliary duct. Hepatic and plasma oxidative stress markers were evaluated by changes in the amount of lipid peroxides, measured as malondialdehyde (MDA) and reduced glutathione (GSH) in plasma and homogenates of hepatic tissue. Serum bilirubin, alkaline phosphatase (AP), and gamma-glutamyl-transpeptidase (GGT) were used to evaluate the severity of cholestasis, and serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were used to evaluate the hepatic injury. Both vitamin E and melatonin were administrated 1 day before and 7 days after bile duct ligation. Acute ligation of the bile duct was accompanied by a significant increased in MDA and a decrease in GSH levels in both plasma and liver, as well as a significant reduction in antioxidant enzymes activities. The overall analysis of both treatments showed that melatonin (500 microg/kg daily) offered significantly better protection against cholestasis and a superior protective effect on hepatic injury than did vitamin E (15 mg/kg daily). Although vitamin E treatment resulted in a reduction of parameters of oxidative stress, the results were significantly better after a much lower daily dose of melatonin. Moreover, melatonin treatment was associated with a significant recovery of antioxidative enzymes. In conclusion, the present paper demonstrates a correlation between the intensity of biliary tract obstruction and increased free radical generation, as well as a direct correlation between the level of oxidative stress and the biochemical markers of liver injury. Melatonin (at a much lower dose than vitamin E) was much more efficient than vitamin E in reducing the negative parameters of cholestasis, the degree of oxidative stress and provided a significantly greater hepatoprotective effect against the liver injury secondary to the acute ligation of the biliary duct.

Melatonin protects against renal oxidative stress after obstructive jaundice in rats.

The goals of this study were to analyze the renal oxidative status in experimental biliary obstruction and to evaluate the impact of melatonin on renal oxidative stress. Cholestasis was done by double ligature and section of the extra-hepatic biliary duct. Melatonin was injected i.p. (500 microg/kg/day). Malondialdehyde, reduced glutathione, catalase, superoxide dismutase, glutathione reductase, glutathione peroxidase and glutathione transferase were determined in the renal tissue. After biliary obstruction, an increase in malondialdehyde (P<0.0001) and a fall in reduced glutathione (P<0.0001) were seen. Moreover, the scavenger enzyme activity had significantly diminished. After melatonin administration, the malondialdehyde fell significantly (P<0.0001), whereas reduced glutathione showed an important increase (P<0.0001) compared with the ligated bile duct group. Experimental bile duct obstruction was associated to an increase of renal oxidative stress. Treatment with melatonin decreased the renal lipid peroxidation, and both the reduced glutathione as well as the scavenger enzyme activity recovered.

Cytokines and acute-phase response markers derangements in patients with obstructive jaundice.

BACKGROUND/AIMS: Prolonged acute-phase response and increase of cytokines have been associated with higher mortality and surgical complications. This study investigated the status of cytokines and acute-phase response markers in patients with obstructive jaundice. METHODOLOGY: Forty-one patients were investigated. Endotoxin, tumor necrosis factor-alpha, interleukin-6, nitric oxide, C-reactive protein, liver enzymes, albumin and percentage of weight loss were determined at admission. RESULTS: Endotoxin, interleukin-6 and C-reactive protein were significantly elevated in both benign and malignant obstructive jaundice. Increased plasma levels of tumor necrosis factor-alpha were only detected in malignant tumors (68 vs. 24 pg/mL; P < 0.001). Patients with positive acute-phase response (C-reactive protein > mean + 2 SD of controls) had greater weight loss (P = 0.02), endotoxin (P = 0.03) and interleukin-6 plasma levels (P = 0.05) than those with no inflammatory response. Prolonged biliary obstruction (> 10 days) was associated with higher weight loss (P = 0.04), tumor necrosis factor-alpha (P = 0.003) and interleukin-6 (P = 0.05) plasma levels. CONCLUSIONS: A prolonged high-grade biliary tract obstruction prompted an increase in endotoxin levels, associated with a positive acute-phase response and cytokine elevation.

Anorexia and the effect of internal biliary drainage on food intake in patients with obstructive jaundice.

BACKGROUND: Anorexia is a frequent finding in patients with biliary obstruction (BO). This study investigates the role of biochemical and hormonal factors in the pathogenesis of reduced food intake in BO and the effects of internal biliary drainage. STUDY DESIGN: Sixty-two patients with BO were prospectively investigated. Transaminases, amylase, cholecystokinin, secretin, bile acids, tumor necrosis factor-alpha, and endotoxin were determined at admission. Caloric intake was quantified by a controlled diet. In a subset of 27 patients, studies were repeated after internal biliary drainage. RESULTS: Sixty-six percent of patients had spontaneous food intakes below the estimated caloric requirements. Serum bilirubin, alkaline phosphatase, and cholecystokinin plasma levels were independent predictor factors for calorie intake (p = 0.0001). After internal biliary drainage, cholestasis parameters and cholecystokinin concentrations decreased significantly; this was associated with an improvement of spontaneous food intake in both benign and malignant biliary obstruction (p < 0.01 and p < 0.05, respectively). CONCLUSIONS: Decreased food intake in BO was associated with the degree of obstruction and with increased cholecystokinin plasma levels. Biliary drainage improved biochemical and food intake derangements.

Factors predicting nutritional derangements in patients with obstructive jaundice: multivariate analysis.

Patients with obstructive jaundice (OJ) that requires surgery often have malnutrition associated with increased perioperative morbidity. This study investigated the factors influencing nutritional derangements in these patients. A series of 46 OJ patients were investigated prospectively (28 malignant tumors, 18 benign obstructions). A nutritional risk index of < 83.5 was used to define protein-calorie malnutrition. Liver function, cholecystokinin (CCK), tumor necrosis factor-alpha (TNFalpha), and endotoxin levels were determined. A multivariate analysis was performed, and an obstructive jaundice malnutrition index (OJMI) was obtained. Altogether, 22 (48%) OJ patients had malnutrition (33% with benign obstructions, 57% with malignant disease). Malnourished patients had higher serum bilirubin levels (258 +/- 120 vs. 154 +/- 62 mmol/L; p = 0.005), longer duration of jaundice (16 +/- 9 vs. 9 +/- 5 days; p = 0.03), and higher plasma levels of CCK (4.0 +/- 1.3 vs. 1.7 +/- 1.0 pmol/L; p = 0.005), alanine aminotransferase (ALT) (226 +/- 209 vs. 187 +/- 161 UI/L; p = 0.01), endotoxin (15 +/- 10 vs. 6.5 +/- 7.0 EU/L; p = 0.007), and TNFalpha (69 +/- 82 vs. 23 +/- 15 pg/ml; p = 0.008) than those without malnutrition. However, only serum bilirubin, CCK, ALT, and patient age were predictors for malnutrition by multivariate analysis. Malnutrition might be expected (95% confidence interval) in patients older than 68 years with increased bilirubin (> 290 mmol/L) and ALT (> 210 UI/L) levels that corresponded with an OJMI > 55. It was concluded that nutritional alterations in patients with obstructive jaundice were determined by the intensity of the biliary obstruction correlated with increased plasma CCK levels as well as with liver dysfunction and patient age.

Changes in the pattern of visceral protein concentrations after internal biliary drainage in patients with obstructive jaundice.

OBJECTIVE: To evaluate the influence of internal drainage on status of nutritional markers in patients with obstructive jaundice. DESSING: Prospective longitudinal study. SETTING: University hospital, Spain. SUBJECTS: 39 patients with obstructive jaundice (18 benign and 21 malignant obstructions). INTERVENTIONS: Nutritional state was assessed before and 10 days after endoscopic drainage. MAIN OUTCOME MEASURES: One anthropometric (body weight <95% of ideal) and two biochemical (albumin <35 g/L and prealbumin < 170 mg/L) as an indication of protein calorie malnutrition. Retinol binding protein and transferrin concentrations, total lymphocyte count, and nutritional prognostic index (NPI) were also measured. RESULTS: Thirty patients (77%) had protein calorie malnutrition. After internal drainage, 6 patients with benign obstruction and 11 with malignant tumours remained malnourished. No anthropometric variables or concentrations of proteins with long half-lives were affected by drainage. However, prealbumin (p < 0.01) and transferrin (p < 0.01) concentrations, and total lymphocyte count (p < 0.001) increased significantly in both groups. NPI also improved significantly after drainage from 43 (9) compared with 37 (5) in benign obstructions (p < 0.05) and 58.7 (14) compared with 52 (12) in malignant (p < 0.05), although in the latter group the mean nutritional risk index remained high. CONCLUSIONS: Concentrations of some of the visceral proteins studied (prealbumin and transferrin) improved 10 days after internal biliary drainage for both benign and malignant obstruction. However, many patients with malignant tumours remained malnourished with a high nutritional risk index.