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.

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.

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.

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.

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.

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.

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.

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.

[Evaluation of anorexia in patients with bile duct obstruction]. / Evaluación de la anorexia en enfermos con obstrucción de la vía biliar.

INTRODUCTION: Obstructive jaundice is often accompanied by protein-caloric malnutrition. The objective of the present study is to analyze the incidence and the degree of alterations in the food ingestion of patients with obstructive jaundice. MATERIAL AND METHODS: In a prospective, cross-sectional study 50 patients with obstructive jaundice (19 benign and 31 malignant) were evaluated. The anorexia was evaluated using Welch's test (subjective evaluation) and by means of quantifying the caloric ingestion. An anthropometric parameter (ideal weigh < 95%) and two biochemical ones (albumin < 3.5 g/dl and pre-albumin < 17 mg/dl) were used to define the degree of malnutrition. RESULTS: 96% of the patients presented alterations in the Welch test and in 72% of the patients the caloric ingestion was below the estimated needs. Overall, the ingestion of food was reduced by 76.3 +/- 30% of the estimated needs (84.7 +/- 28% in the benign cases and 70.9 +/- 32% in the malignant cases). Both the Welch test (r = 0.59; p = 0.01) and the caloric ingestion (r = 0.53; p < 0.001) were inversely correlated with the serum bilirubin. In patients with malnutrition criteria, the caloric ingestion was reduced by 30% against the 12% reduction in the non-malnourished patients (p < 0.05). There was a direct correlation between the two methods used in the assessment of the anorexia (r = 0.71; p < 0.001). CONCLUSIONS: Obstructive jaundice is associated with an important reduction in the caloric ingestion, and this is manifested in both biliary obstructions of a benign origin, and in those of neoplasic origins.

[The results of duodenopancreatectomy in periampullar pathology]. / Resultados de la duodenopancreatectomía cefálica en patología periampular.

OBJECTIVE: To examine mortality and morbidity rates after pancreaticoduodenectomy in 69 consecutive patients with periampullar disease operated on between 1985 and 1993 at the Reina Sofía Hospital, Córdoba, Spain. PATIENTS: Fifty five patients (79.7%) had malignant neoplasm whereas 14 (20.3%) had benign disease. In 58 patients a Whipple procedure was performed; pancreaticoduodenectomy with preservation of the pylorus (Traverso-Longmire) was performed in the remaining 11 patients. RESULTS: Eighteen patients (26%) had postoperative complications; peritoneal bleeding (1); biliary fistula (3); pancreatic fistula (4); digestive fistula (2); and pancreatitis (2). Two patients with pancreatic and duodenal carcinoma died. Thirteen patients were readmitted: 3 bleeding episodes in anastomotic ulcer; 3 hepaticojejunostomy obstruction; and the remaining 6 patients with tumor recurrence. CONCLUSIONS: In our experience duodenopancreatectomy was a safe procedure in periampullar disease, with an acceptable morbidity and mortality rates.

[Prognostic factors in severe acute pancreatitis. Univariate and multivariate study]. / Factores pronósticos en la pancreatitis aguda severa. Estudio univariante y multivariante.

OBJECTIVES: The aim of this study was to analyze the prediction of mortality in patients with severe acute pancreatitis. PATIENTS AND METHODS: In a retrospective study 43 patients with severe acute pancreatitis were included. All patients required ICU admittance and surgical treatment. We evaluated severity according to Ranson's criteria, APACHE II score, CT scan classification (Hill), intraoperative findings (Vankemmel's classification) and number of organs failure. We performed a univariant and multivariant statistic study with lineal discriminant analysis. RESULTS: The overall mortality was 46.5%. Ranson's score and APACHE II did not correlate with mortality. Hill's classification did not reach significance either. However, only the Vankemmel's classification and the number of organs failure had statistic value (p < 0.01). After lineal discriminant analysis, the association of more than 4 Ranson's criteria, APACHE II up to 9 points, grades IV and V in Hill's classification and 4 organs failure had a predictive value for mortality. CONCLUSIONS: In our limited experience the Vankemmel's classification and the number of organs failure had a predictive value for mortality in patients with acute pancreatitis. The association of more than 4 Ranson's criteria. APACHE II up to 8 points, grades IV-V in Hill's classification and 4 organs failure disclosed poor prognosis.

[The palliative treatment of pancreatic cancer: percutaneous drainage versus surgical diversion]. / Tratamiento paliativo del cáncer de páncreas: drenaje percutáneo versus derivación quirúrgica.

In a retrospective study, the outcome of 56 patients with unresectable carcinoma of the exocrine pancreas undergoing two palliative operations (bypass surgery vs percutaneous transhepatic biliary drainage with endoprosthesis) for the management of biliary obstruction was evaluated. Morbidity and mortality were similar in the two groups. Postop hospital stay was 8 days for the intubated, and 15 days for operated patients (p less than 0.01). Although the symptom-free period was similar in both groups, intubated patients had a shorter period of poor-quality life.

[Hepatic confluence tumors: results and quality of life according to the type of treatment]. / Tumores de la confluencia hepática: resultados y calidad de vida según el tipo de tratamiento.

We analyzed survival rate and quality of life according to the treatment modality in 52 patients with hilar tumors. Six of them underwent resection (11.5%); 4 without hepatectomy, one left hepatectomy and one trisegmentectomy. The remaining 46 patients were treated by: radiologic external-internal drainage (12), placement of percutaneous endoprostheses (14), surgical intubation (18), and cholangioanastomoses to segment III in 2. Prognostic factors (PITT and A.P.A.C.H.E. II), survival time and quality of life were analyzed. Survival and comfort index were significantly better (p < 0.001) in the resection group than in the palliation one. Among palliative procedures percutaneous endoprostheses and surgical intubation offered better quality of life (p < 0.001) than radiologic external-internal drainage. We conclude that resection improves survival and offers better quality of life than palliative procedures. Our results suggest that resection during laparotomy should be attempted in order to improve results in the treatment of hepatic confluence tumors.

[Self-expanding metallic endoprostheses in the palliative treatment of biliary obstructions in nonresectable carcinoma of the head of the pancreas]. / Endoprótesis metálicas autoexpansivas en el tratamiento paliativo de la ictericia obstructiva en el carcinoma de cabeza de páncreas irresecable.

OBJECTIVE: A study of immediate and long-term results with the self-expandable metallic stent (Wallstent), in the treatment of biliary obstruction in 25 patients with non resectable carcinoma of the head of the pancreas was carried out. Stent placement was successful in all patients. RESULTS: Complication rate was 4% (n = 1); one patient had venous bleeding after percutaneous catheter placement. There was no procedure related mortality (30-day mortality); hospital stay was 6, 7 days (2-12). Average survival was 6 months (+/- 2.97). Average patency of the initial stent lasted 5 months (+/- 2.01); comfort index was 83%. Five patients required re-admission. Late complications were cholangitis in 2 and stent occlusion in 4. Disimpaction in one patient and placement of additional stent (PAL-MAZ) in the remaining 3 patients were performed. One patient required surgical treatment; hepaticojejunostomy was performed. Elapse time between prostheses placement and stent occlusion was 3.4 months (2-4.5). CONCLUSIONS: We conclude that metallic stent placement has low morbidity without mortality and provide good quality of live. The most frequent late complication was prostheses obstruction.

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.