Serum levels of interleukin-9 during acute rejection in liver transplantation.

INTRODUCTION: Interleukin-9 (IL-9) has been cast as a player in autoimmunity, but its role in liver transplantation remains to be clarified. The aim of our study was to investigate the time course of IL-9 serum levels during hepatic allograft rejection. METHODS: IL-9 serum levels were determined in 34 healthy subjects and 50 hepatic transplant recipients. The patients were divided into two groups: group I was composed of 15 patients with acute rejection episodes, and group II, 35 patients free of this problem. Samples were collected on days 1 and 7 after liver transplantation and on the day of liver biopsy. RESULTS: The concentrations of IL-9 were similar in the rejection and nonrejection groups over the entire postoperative period. The whole transplant group, including those with stable graft function, showed higher IL-9 serum levels than the controls at all times after liver transplantation. CONCLUSIONS: These preliminary results suggest a lack of participation of IL-9 in human liver allograft rejection.

[Gene expression in obese patients with non-alcoholic steatohepatitis]. / Expresión génica en pacientes obesos con enfermedad hepática por depósito de grasa.

UNLABELLED: Although the molecular basis for the pathophysiology of non-alcoholic steatohepatitis (NASH) is poorly understood, we evaluate the hepatic gene expression of cytokines, chemokines, cell receptors, growth factors, intracellular transducers and extracellular communication proteins in liver tissue of obese patients (with and without NASH), and we determine the specific intrahepatic gene expression profiles associated with histological severe NASH.Thirty-eight obese patients with BMI > 35 were analyzed, who underwent bariatric surgery. Biopsy specimen samples were snap-frozen in liquid nitrogen. Hepatic gene expression was determined in liver biopsy specimens from 3 groups: a) obese patients without NASH (n = 12); b) patients with NASH without fibrosis (n = 13); and c) patients with NASH and fibrosis (n = 13). Genes were considered to be expressed differentially in NASH only if there was a greater than 2-fold difference in abundance of mRNA when compared with each of the control group. These results were confirmed by real-time PCR. Fourteen genes were differentially expressed (10 overexpressed and 4 underexpressed) in patients with NASH. Genes that were significantly overexpressed included prohibitin, TNF, TNF RI (p55), MCSF, R2-TRAIL, b1-CTGF, FGF, VEGF, and BIGH3OBR. Insulin growth factor-1, insulin growth factor-2, interleukin-2 and tyrosine-receptor were underexpressed in NASH patients. IN CONCLUSION: 1. The obese patients with NASH without fibrosis show an overexpression of proinflammatory and proapoptotic genes. Also, the NASH patients with fibrosis show an overexpression of fibrogenic genes, including the leptin receptor Ob-Rb.2. The up-regulated gene expression of prohibitin suggests mitochondrial dysfunction in NASH patients.

Adiponectin and its receptors in non-alcoholic steatohepatitis.

BACKGROUND: Adiponectin, an adipocyte derived polypeptide, has been shown to alleviate steatosis and inflammation in mice with non-alcoholic fatty liver disease. AIM: In the present study, we wished to define liver expression of adiponectin and its receptors in morbidly obese patients undergoing bariatric surgery. Patients with non-alcoholic steatohepatitis (NASH) or simple steatosis were investigated to test whether dysregulation of this system might be involved in these disorders. PATIENTS AND METHODS: Liver mRNA expression of adiponectin and its recently cloned receptors RI and RII (adipoRI and adipoRII) were analysed by fluorescence based real time polymerase chain reaction in 13 patients with NASH and nine with simple steatosis. Adiponectin and adipoRII protein expression were assessed by immunohistochemistry in a subgroup of patients. RESULTS: Adiponectin and adipoRII mRNA expression were significantly reduced in liver biopsies of patients with NASH compared with simple steatosis while no difference was found in adipoRI mRNA expression. In NASH, adipoRII mRNA expression was negatively correlated with serum aspartate aminotransferase levels, serum alanine aminotransferase levels, and grade of fibrosis. Liver adiponectin protein expression was mainly found in endothelial cells of portal vessels and liver sinusoids whereas adipoRII expression was seen in hepatocytes only. Adiponectin and adipoRII staining were lower in biopsies of subjects with NASH compared with simple steatosis. CONCLUSION: Reduced hepatic expression of adiponectin and adipoRII might be of pathophysiological relevance in non-alcoholic fatty liver diseases.

[Triple immunosuppressive therapy in the treatment of severe ulcerative colitis]. / Triple terapia inmunosupresora en el tratamiento de la colitis ulcerosa grave.

INTRODUCTION: Severe episodes of steroid-refractory ulcerative colitis (UC) were considered an indication for surgery until the introduction of new immunosuppressive agents such as cyclosporine. OBJECTIVES: 1) To confirm the efficacy of intravenous cyclosporine in inducing remission in severe episodes of steroid-refractory UC; 2) To analyze the efficacy of triple immunosuppressive therapy with cyclosporine, azathioprine and prednisone in the maintenance of remission induced by intravenous cyclosporine. PATIENTS AND METHOD: Fourteen patients diagnosed with a severe episode of steroid-refractory UC were treated with intravenous cyclosporine at a dose of 4 mg/kg/day. In all patients, after response was induced, this regimen was substituted by oral cyclosporine plus azathioprine at a dose of 2-2.5 mg/kg/day and decreasing doses of corticoids. Neoral cyclosporine was progressively reduced until discontinuation within 3 months, coinciding with a simultaneous decrease of oral steroids. RESULTS: All patients showed response to intravenous cyclosporine with a significant reduction in the Truelove index calculated before and after treatment. After remission was induced, all patients followed triple immunosuppressive therapy for 3 months. In the follow-up for a mean of 24 months (range: 14-34) only two patients required admission for a new episode of UC and colectomy was finally indicated in only one. None of the 14 patients treated with cyclosporine showed severe adverse effects attributable to the drug. CONCLUSIONS: Intravenous cyclosporine is a safe and effective alternative in the treatment of severe episodes of steroid-refractory UC. Early initiation of oral administration associated with azathioprine is useful in maintaining response, reducing subsequent relapses and the need for colectomy during the follow-up of these patients.

[Plasma leptin levels in patients with primary biliary cirrhosis and their relationship with degree of fibrosis]. / Concentraciones plasmáticas de leptina en los pacientes con cirrosis biliar primaria y su relación con el grado de fibrosis.

OBJECTIVES: a) To analyze plasma leptin levels in patients with primary biliary cirrhosis (PBC) and b) to investigate the relationship between leptin levels and liver fibrosis stage in a cohort of patients with PBC. PATIENTS AND METHODS: Serum leptin levels were evaluated through radioimmunoassay in 30 patients with PBC (mean age: 37.2 +/- 11.0 years; range:19-75) and in 29 controls matched for age and weight. Venous blood obtained after a 12-hour fast was centrifuged in EDTA tubes. Weight, height and body mass index (BMI) were measured using standard methods. Hepatitis C virus RNA was determined using qualitative and quantitative polymerase chain reaction. In all patients liver biopsies were performed and the degree of fibrosis and extent of inflammatory infiltrate were evaluated. RESULTS: Plasma leptin levels in patients with PBC were lower than those obtained in control subjects (p<0.0001). No significant differences were found between the two groups in age, weight, height, BMI or body fat index. There was a clear increase in serum leptin levels according to histological stage of PBC (stage I: 2.1 ng/ml; stage II: 4.3 ng/ml; stage III: 5.3 ng/ml; stage IV: 12.1 ng/ml; p<0.01) CONCLUSIONS: The present study demonstrates the correlation between leptin and stage of liver fibrosis in a cohort of patients with PBC, providing further evidence of the involvement of leptin in the process of liver fibrosis.

Involvement of the Fas system in liver allograft rejection.

OBJECTIVE: Recent studies suggest that apoptosis is an important mechanism of cell death in the rejection of liver allografts and that this process is mediated via Fas. The aim of this study was to analyze the expression of the Fas system during the liver allograft rejection and its evolution after treatment. METHODS: We evaluated 14 patients with liver allograft rejection before and after treatment. Fas immunostaining was performed by the labeled streptavidin-biotin peroxidase method using a 200-fold dilution of a monoclonal antibody. Assessment of apoptosis was determined by the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) technique on deparaffined liver samples. Serum levels of soluble Fas antigen (sFas) were detected by an enzyme immunoassay procedure. Twelve liver transplant patients without allograft rejection were analyzed as a control group. RESULTS: The number of hepatocytes expressing Fas antigen, the percentage of apoptotic hepatocytes, and the sFas levels were higher in patients with liver allograft rejection than in controls (27.9+/-23.1% vs 1.4+/-1.2%, p < 0.001; 2.2+/-0.9% vs 1.0+/-0.1%, p = 0.02; 24.2+/-39.6 vs 2.8+/-4.0 IU/ml, p = 0.03, respectively). There was a correlation between the levels of sFas, AST (r = 0.86, p < 0.001), ALT (r = 0.78, p = 0.02), and gamma-globulin levels (r = 0.86, p < 0.001). After the rejection treatment we found a significant decrease in the Fas antigen expression (18.6+/-13.3%, p < 0.05), TUNEL index (0.2+/-0.4, p < 0.05), and levels of sFas (9.9+/-30.25 IU/ml, p = 0.005). CONCLUSIONS: 1) The demonstration of hepatocytes with Fas antigen expression and the labeling of the nuclei by the TUNEL assay suggest that apoptosis mediated by the Fas system plays a role in the pathogenesis of liver allograft rejection. 2) The Fas expression and the sFas levels decreased in patients with treatment response.

Apoptosis mediated by the Fas system in the fulminant hepatitis by hepatitis B virus.

The pathogenesis of the fulminant hepatitis B is poorly understood and both viral factors and the hosts immune response play a role. Previous studies in liver tissues of patients with chronic hepatitis B showed overexpression of Fas antigen and this was correlated with the activity of the hepatitis. The present study was done to determine the role of Fas in fulminant hepatitis B and the virological characteristics of hepatitis B infection. We studied three patients with fulminant hepatitis B. HBV-DNA was detected by dot-blot hybridization and polymerase chain reaction. The S and C gene were sequenced. Levels of serum soluble Fas antigen were detected by enzymoimmunoassays procedure. Apoptosis was determined by the TUNEL technique. Fas antigen expression was evaluated by a immunoperoxidase method. Ten healthy subjects acted as controls. The three patients showed a high expression of Fas antigen particularly among infiltrating lymphocytes; in these areas we also found many cells with in situ DNA nick labelling signals in the nuclei of most viable hepatocytes. Serum levels of soluble Fas antigen were higher in patients with fulminant hepatitis B than in controls. No specific genome mutations of hepatitis B virus were found. These data suggest that the Fas system involved in the liver injury of patients with fulminant hepatitis B.

Gene expression of tumor necrosis factor alpha and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients.

The main objective of this study was to analyze the pathogenic role of the tumor necrosis factor alpha (TNF-alpha) system in the development of nonalcoholic steatohepatitis (NASH). Fifty-two obese patients were studied. We investigated: (1) the expression of mRNA of TNF-alpha and their p55 and p75-receptors by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) in hepatic and adipose tissues; and (2) the relationship between TNF-alpha, p55, and p75 and the severity of NASH. Obese patients without NASH were the control group. A remarkable increase in the expression of mRNA of TNF-alpha was found in patients with NASH in hepatic tissue (0.65 +/- 0.54) and in peripheral fat (0.43 +/- 0.45); in the control samples, the mRNA expression was 0.28 +/- 0.32, P <.007, and 0.26 +/- 0.22, P <.018, respectively. Furthermore, we found a significant increase in the mRNA levels of p55 receptor (2.42 +/- 1.81 vs. 1.56 +/- 1.17; P <.05); however, the mRNA expression of the p75 receptor was similar in both patients. Those patients with NASH with significant fibrosis presented an increase in the expression of mRNA TNF-alpha in comparison with those with a slight or nonexistent fibrosis. An overexpression of TNF-alpha mRNA is found in the liver and in the adipose tissue of NASH patients. The levels of mRNA-p55 are increased in the liver tissue of NASH patients. This overexpression is more elevated in patients with more advanced NASH. These findings suggest that the TNF-alpha system may be involved in the pathogenesis of NASH.

Dendroaspis natriuretic peptide in hepatic cirrhosis.

OBJECTIVES: Dendroaspis natriuretic peptide (DNP) is a novel peptide that is structurally similar to atrial, brain, and C-type natriuretic peptides. Many natriuretic peptides are increased in hepatic cirrhosis, but the role of DNP in cirrhosis is unknown at present. The aim of the study was to investigate plasma levels of dendroaspis natriuretic-like immunoreactivity in cirrhosis. METHODS: We measured plasma concentrations of DNP by radioimmunoassay methods in 12 cirrhotic patients without ascites and 44 cirrhotic patients with ascites, and compared these values with 20 age-matched healthy subjects. Renal function, plasma cGMP concentration, plasma renin activity, and plasma endothelin concentration were measured in each patient. RESULTS: Patients without ascites had circulating levels of DNP similar to those of healthy subjects. By contrast, patients with ascites had increased circulating DNP levels compared to both patients without ascites and healthy subjects. In addition, circulating levels of DNP increased in relation to the severity of cirrhosis. Significant positive correlations were also found between DNP levels, endothelin concentrations, and plasma renin activity. CONCLUSIONS: The results of this study indicate that plasma DNP is increased in cirrhotic patients with ascites.

Are there predictive factors of severe liver fibrosis in morbidly obese patients with non-alcoholic steatohepatitis?

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a clinicopathological entity characterized by the presence of steatosis and lobular and/or portal inflammation with or without fibrosis. Patients with non-alcoholic fatty liver and fibrosis on liver biopsy have increased liver-related deaths. METHODS: 181 wedge liver biopsies, taken at the time of bariatric surgery from patients with a mean body mass index (BMI) of 47, were studied. In all cases, the liver biopsy was performed without knowledge of the patient's clinical and biochemical data, which were then examined with univariate and multivariate analysis. RESULTS: Diagnosis of NASH was established in 105 patients (91%); 74 patients (70%) showed mild steatosis, 20 (19%) had moderate inflammation and fibrosis, and 11 (10%) had steatosis with severe fibrosis. None of the liver biopsies showed cirrhosis. Age was the only independent predictor of moderate and severe fibrosis (p = 0.001). CONCLUSIONS: Since only age was a predictor of moderate or severe fibrosis, and no clinical or biochemical abnormalities detected slowly progressive hepatic fibrosis, liver biopsy is the only means of detecting progression to more advanced liver disease in a NASH patient.

Involvement of the fas system in hepatitis C virus recurrence after liver transplantation.

To date, there have been no reports of the involvement of the Fas system in recurrent hepatitis C virus (HCV) infection after orthotopic liver transplantation (OLT). In 25 patients who underwent OLT for HCV-related liver cirrhosis, we evaluated the expression of the Fas antigen (FasAg) on hepatocytes, apoptic hepatocytes, and serum levels of soluble Fas (sFas). The level of HCV viremia and HCV genotype were determined by polymerase chain reaction. Serum sFas levels were determined by an enzyme immunoassay procedure. DNA fragmentation was determined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) technique on deparaffinized liver samples. FasAg expression was evaluated by an immunoperoxidase method. Sixteen patients had evidence of recurrent HCV disease. The number of hepatocytes expressing FasAg and the percentage of apoptotic hepatocytes was greater among patients who developed recurrent hepatitis than among those who did not (P <.01 and P <.0001, respectively). There was a correlation between hepatic expression of FasAg, intensity of lobular inflammation (P =.007), and TUNEL index (P <.001). The levels of sFas were greater among the patients with recurrent HCV hepatitis than those without recurrent hepatitis (P <.04). We conclude that (1) Fas expression is up-regulated in recurrent HCV after OLT and is related to the grading of liver disease; likewise, levels of sFas were greater in the patients with recurrent HCV hepatitis; and (2) the demonstration of hepatocytes with FasAg expression and the labeling of the nuclei by TUNEL assay suggest that hepatic apoptosis mediated by the Fas system may have a role in the pathogenesis of recurrent HCV hepatitis after OLT.

[Soluble Fas in acute rejection in patients with liver transplantation. Response to treatment]. / Fas soluble en el rechazo agudo de los pacientes con trasplante hepático. Respuesta al tratamiento.

UNLABELLED: It has been suggested that in the cellular rejection of hepatic graft, the lesion provoked by cytotoxic T-lymphocytes might be mediated by the Fas antigen/Fas ligand system, producing cell death by apoptosis. AIM: To determine whether soluble Fas (sFas) is increased during treatment and to evaluate response to treatment. MATERIAL AND METHODS: Fourteen patients with orthotopic liver transplantation who presented an episode of acute cellular rejection were studied. Serum levels of sFas were detected by enzyme-immunoanalysis at diagnosis and again 5 days after treatment was completed. As control groups, sFas was analyzed in 12 patients with liver transplantation without rejection and in 10 healthy subjects. RESULTS: sFas levels were found to be significantly higher in patients with rejection than in those without rejection and in healthy subjects (24.2 +/- 39.1 vs. 2.8 +/- 4.0 vs. 0.6 +/- 1.5 UI/ml; p = 0.03). In patients with acute cellular rejection, immunosuppressive treatment significantly decreased sFas levels compared with basal values (24.2 +/- 39.1 vs. 9.9 +/- 30.2 UI/ml; p = 0.005). A clear correlation between the serum levels of sFas, glutamate oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase/glutamic-pyruvic transaminase (GPT) and gamma-glutamyl transferase (GGT). CONCLUSIONS: a) The increase of sFas during rejection and the decrease in patients with a good evolution suggests that the Fas system may play an important role in the hepatocyte lesion produced by an apoptosis mechanism in these patients. b) Monitoring sFas could be useful in diagnosing rejection and in evaluating response to treatment.

Different time course of circulating adhesion molecules and hyaluran during hepatic allograft rejection.

BACKGROUND: Inducible adhesion molecules are involved in cell-mediated allograft rejection. In addition, the endothelium is the main target of this process. This study investigated, whether soluble (s) forms of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) are elevated during cellular rejection and whether hyaluran is a useful marker of endothelial function in liver transplantation. METHODS: Serum levels of sICAM-1, sVCAM-1, and hyaluran were determined in 24 controls and 27 hepatic transplant recipients. These patients were divided in two groups: group I, 14 patients without rejection; and group II, 13 patients with rejection. Samples were collected on day 1 and 7 after transplantation, on the day of liver biopsy, and after treatment of the rejection. RESULTS: We found a significant increase in sICAM-1 levels in the postoperative period in the rejection group compared with the non rejection group. It persisted significantly elevated until the diagnosis of rejection was made. In contrast, sVCAM-1 was only significantly elevated in the rejection group when diagnosis of rejection was evident. Hyaluran levels were also significantly elevated in the rejection group at diagnosis of rejection. We noticed a significant decline in sICAM-1, sVCAM-1, and hyaluran levels after successful treatment of rejection. In addition, we observed in the non-rejection group a stable lower levels of hyaluran during the entire postoperative period. CONCLUSIONS: The release of circulating adhesion molecules is a prominent feature coinciding with the first episode of hepatic rejection. Differential patterns of sICAM-1 and sVCAM-1 exist during rejection. In addition, hyaluran levels may be a sensitive marker of liver endothelial cell function in the postoperative period of liver transplantation.

Adrenomedullin in liver transplantation and its relationship with vascular complications.

Adrenomedullin (AM) is a potent vasodilating peptide that increases rat platelet cyclic adenosine monophosphate levels and acts on endothelial cells to stimulate nitric oxide release. Both mechanisms inhibit platelet function. Considering these effects, AM may have a role in cardiovascular regulation after orthotopic liver transplantation (OLT) and could have an antithrombotic effect. The aim of the present study is to investigate plasma AM levels in the early postoperative period after OLT and their relationship with vascular complications in OLT. We measured plasma AM levels in 35 patients with cirrhosis who underwent OLT at baseline and 1, 7, and 15 days postoperatively. We found that AM levels were significantly greater in patients with cirrhosis compared with healthy subjects. Of the 35 patients, 10 had vascular complications. In these 10 patients, AM concentrations were significantly greater than those observed in the nonthrombotic group in the early postoperative period. In addition, we also noticed in the nonthrombotic group a significant increase in AM levels from baseline to day 1, then a decrease to baseline levels in the early postoperative period. Our study shows that AM might act as a new humoral factor involved in the response to surgery in OLT and is significantly associated with vascular thrombosis in OLT.

[The prevalence of the Cys282Tyr mutation in the hemochromatosis gene in Cantabria in patients diagnosed with hereditary hemochromatosis]. / Prevalencia de la mutación Cys282Tyr del gen de la hemocromatosis en Cantabria y en los pacientes diagnosticados de hemocromatosis hereditaria.

BACKGROUND: The aim of our study was to evaluate the prevalence of Cys282Tyr mutation in patients with genetic haemochromatosis (GH) in Cantabria. PATIENTS AND METHODS: The HFE Cys282Tyr mutation was determined in a cohort of 60 patients with GH and 213 controls. RESULTS: The frequency of the Cys282Tyr mutation in control individuals was 4.4%. Sixty-seven percent of patients with GH were homozygous for the Cys282Tyr mutation. Twenty-seven percent of patients were normal at Cys282Tyr loci. CONCLUSIONS: The prevalence of the Cys282Tyr mutation in patients with GH in Cantabria, Spain, seems to be lower than in North America and in North Europe patients.

Severe clinical course of de novo hepatitis B infection after liver transplantation.

The aim of this study is to determine the origin, clinical outcome, allograft histological characteristics, and virological outcome of de novo hepatitis B virus (HBV) infection after orthotopic liver transplantation (OLT). We studied 136 hepatitis B surface antigen (HBsAg)-negative liver transplant recipients. HBV DNA was detected by dot-blot hybridization and polymerase chain reaction (PCR). The S gene was sequenced. Hepatitis C virus (HCV) RNA was assessed by PCR. The long-term clinical and histological outcome was determined. Six of 136 HBsAg-negative patients (4.4%) became HBsAg positive after transplantation. The source of HBV infection was reactivation of latent HBV infection in 2 patients and was not identified in 4 patients. Two donors had isolated core antibody. Two of these 6 patients developed acute liver failure related to hepatitis B. The 4 other patients had severe chronic hepatitis related to hepatitis B. All patients had high-level HBV replication. No significant mutations in the S gene were found. These data suggest that de novo hepatitis B infection is not a mild disease and might represent a significant cause of graft dysfunction. This is the first report of fulminant hepatitis caused by de novo hepatitis B infection after OLT.

Plasma adrenomedullin levels in patients with hepatic cirrhosis.

OBJECTIVE: The role of adrenomedullin in cirrhosis is at present unknown. The aim of this study was to investigate plasma levels of adrenomedullin in cirrhosis. METHODS: We measured plasma adrenomedullin concentrations using radioimmunoassay in 43 patients with cirrhosis (17 in Child-Pugh's class A, 13 in Child-Pugh's class B, and 13 in Child-Pugh's class C) and compared these values with those in 24 age-matched healthy subjects. Renal function, plasma cyclic adenosine monophosphate (cAMP) concentration, plasma renin activity, and plasma aldosterone concentration were measured in each patient. RESULTS: Plasma adrenomedullin levels were increased significantly in patients with cirrhosis compared with healthy subjects. In addition, plasma adrenomedullin values were higher in patients with cirrhosis and ascites than in those without ascites. Moreover, plasma adrenomedullin levels increased in relation to the severity of cirrhosis. Significant positive correlations also were found between adrenomedullin and cAMP concentrations and plasma renin activity or plasma aldosterone concentrations. CONCLUSIONS: Plasma adrenomedullin is increased in patients with cirrhosis compared with healthy subjects. The increase in plasma adrenomedullin levels is related at least in part to the severity of cirrhosis and may contribute to the arteriolar vasodilation in patients with cirrhosis. These effects are related in part to a cAMP-dependent pathway.