Lorenz Theilmann

Manganese and chronic hepatic encephalopathy

Clinical observations and animal studies have raised the hypothesis that increased concentrations of manganese (Mn) in whole blood might lead to accumulation of this metal within the basal ganglia in patients with end-stage liver disease. We studied ten patients with liver failure (and ten controls) by magnetic resonance imaging (MRI) and measurement of Mn in brain tissue of three patients who died of progressive liver failure (and three controls) was also done. Whole blood Mn concentrations in patients with liver cirrhosis were significantly increased (median 34.4 micrograms/L vs 10.3 micrograms/L in controls; p = 0.0004) and pallidal signal intensity indices correlated with blood Mn (Rs = 0.8, p = 0.0058). Brain tissue samples reveal highest Mn concentrations in the caudate nucleus, followed by the quadrigeminal plate and globus pallidus. Mn accumulates within the basal ganglia in liver cirrhosis. Similarities between Mn neurotoxicity and chronic hepatic encephalopathy suggest that this metal may have a role in the pathogenesis of chronic hepatic encephalopathy. Further studies are warranted because the use of chelating agents could prove to be a new therapeutic option to prevent or reverse this neuropsychiatric syndrome.

Peripheral blood leukocytes serve as a possible extrahepatic site for hepatitis C virus replication

To study possible extrahepatic sites for the replication of hepatitis C virus (HCV), we examined fresh and cultured peripheral blood mononuclear leukocytes (PBML), as well as different subpopulations of PBML of HCV-infected patients, for the presence of viral genomic and antigenomic RNA. Sense and antisense oligonucleotide primers derived from HCV sequences were used for reverse transcription (RT) followed by an amplification with the polymerase chain reaction assay (PCR). Using antisense primers for RT, genomic viral RNA could be detected in serum, liver, total PBML and B lymphocytes of chronically infected patients. However, only liver tissue and PBML specimens were positive when a sense primer was used. To demonstrate further the specificity of these findings, total PBML were stimulated using pokeweed mitogen and synthesis of HCV RNA was determined by incorporation of [3H]uridine into nascent viral RNA molecules using a hybrid release assay. Additionally, total PBML from an uninfected person could be infected in vitro using an HCV RNA-positive serum. The PCR products obtained from serum, liver and PBML specimens of an HCV-positive individual were found to have nearly identical sequences. Our findings suggest that PBML could be a site for viral replication of HCV during the natural course of infection and may represent a reservoir for hepatitis C virions.

Frequency and predictors of osteoporotic fractures after cardiac or liver transplantation: a follow-up study

BACKGROUND Osteoporosis and related fractures are a major complication after organ transplantation. The aim of this study was to find out the frequency and predictors of osteoporotic fractures after cardiac or liver transplantation. METHODS 235 consecutive patients who had a cardiac transplant (n=105; 88 men, 17 women) or a liver transplant (130; 75 men, 55 women) were followed. Vertebral fractures were assessed by a standardised analysis of spinal radiographs before and annually after transplantation. Clinical and non-vertebral fracture data were noted from hospital records. FINDINGS In the first and second years after transplantation, the proportion of patients (Kaplan-Meier estimates) who had at least one vertebral fracture was slightly higher in the cardiac group (first year 21%, second year 27%) than in the liver group (first year 14%, second year 21%). In the third and fourth years, one third of patients from both groups had had one or more vertebral fractures. Non-vertebral fractures occurred in nine patients (7%) after liver transplantation and avascular necrosis of the hip head in three patients (3%) after cardiac transplantation. In both groups, no dose-dependent effect of immunosuppressive therapy on fracture development could be identified. Independent predictors assessed by multivariate analysis were age (hazard ratio [95% CI] increase of 5 years, 1.71 [1.1-2.7]) and lumbar bone-mineral density (decrease of 1 SD t score, 1.97 [1.2-3.2]) in cardiac transplantation patients, and vertebral fractures before transplantation (6.07 [1.7-21.7]) in the liver group. INTERPRETATION The high frequency of osteoporotic fractures in the 2 years after transplantation and the limitations of reliable fracture-risk predictions, show the need to investigate preventive therapies.

Efficacy of ursodeoxycholic acid treatment and endoscopic dilation of major duct stenoses in primary sclerosing cholangitis: An 8-year prospective study

BACKGROUND/AIMS Primary sclerosing cholangitis is characterized by progressive fibrotic inflammation and obliteration of intra- and extrahepatic bile ducts. Ursodeoxycholic acid treatment leads to improvement of biochemical parameters of cholestasis and in part also of liver histology. During treatment, obstruction of major ducts may lead to deterioration of liver function, which may be prevented by endoscopic dilation of the stenoses. Controlled trials for evaluation of the beneficial effects of ursodeoxycholic acid treatment and of endoscopic measures in patients with major duct stenoses have become clinically difficult. Estimation of survival probabilities without treatment allows comparison of actuarial survival with the estimated survival probabilities. METHODS/RESULTS We studied survival in 65 patients with PSC treated with ursodeoxycholic acid (750 mg/day) and by endoscopic measures whenever necessary. Patients with decompensated cirrhosis in whom transplantation was foreseen were excluded. The study was started in May 1987 and the mean follow-up period was 45.0+/-3.5 (mean+/-SEM) months. Liver histology was performed in each of the patients before entry into the study and revealed that 21% were in stage 1, 37% in stage 2, 21% in stage 3 and 20% in stage 4. Of 65 patients, 12 had major duct stenosis at entry and another 11 developed major duct stenosis during ursodeoxycholic acid treatment, which was successfully treated by repeated endoscopic balloon dilations. The actuarial Kaplan-Meier survival probabilities without liver transplantation after treatment with ursodeoxycholic acid and dilation of major duct stenoses were significantly improved compared to the predicted survival rates with p=0.001. CONCLUSIONS Ursodeoxycholic acid does not prevent major bile duct occlusion. When ursodeoxycholic acid treatment and endoscopic opening of duct stenoses are combined, survival may be significantly improved.

Establishment of persistent hepatitis C virus infection and replication in vitro.

Hepatitis C virus (HCV) is a major cause of chronic viral hepatitis. Development of anti-viral strategies has been hampered by the lack of efficient cell systems to propagate HCV in vitro. To establish a long-term culture system, we tested human hepatoma (HuH7, HepG2) and porcine non-hepatoma (PK15, STE) cell lines, as well as several culture and infection conditions. As a marker for virus replication, minus-strand HCV RNA in infected cells was detected by an enhanced detection system using nested RT-PCR followed by hybridization analysis. Short-term efficiency of HCV infection (10 days) was slightly increased by addition of polyethylene glycol (PEG) and/or dimethyl sulfoxide (DMSO) to culture media during inoculation of HuH7, PK15 and STE cells, but no augmentation in long-term culture was achieved, suggesting enhanced attachment of HCV to cells rather than more efficient infection. A stabilizing effect on HCV propagation was observed for 50 days in a serum-free medium with stimulation of the low-density lipoprotein (LDL) receptor expression by lovastatin. Using partially serum-free culture conditions, long-term persistence of HCV in cells and release of virions into supernatant was achieved for up to 130 days. Infectivity of released virions in supernatants after long-term culturing (day 30-80) was shown by successful infection of fresh cells. In conclusion, supplementation with PEG, DMSO and lovastatin during inoculation did not enhance virus replication substantially, but continued stimulation of LDL-receptor expression resulted in infections which persisted for over 4 months. These data support the hypothesis of an LDL-receptor mediated uptake of HCV into cells in vitro.

Phase-I trial of intravenous continuous infusion of tumor necrosis factor in advanced metastatic carcinomas

SummaryFifteen patients with advanced metastatic adenocarcinomas were treated in a phase-I study with continuous intravenous 24h infusion of recombinant tumor necrosis factor α (TNF-α) in order to determine the maximum tolerated dose (MTD) and associated side-effects. Patients received 40–400 μg/m2 TNF-α once (arm A) or twice (arm B) weekly for a scheduled treatment period of 2 months. The observed systemic side-effects resembled those reported for interferons and included fever, chills, fatigue, headaches, myalgias, thrombocytopenia, prostration, and malaise. Dose-limiting toxicities, resulting in a median MTD of 200 μg/m2 for 24h, were fever, chills, fatique, myalgias, and thrombocytopenia. Out of 15 patients, 11 showed tumor progression, and 3 sustained in no change for over 2 months of treatment. A minor response was seen in 1 patient with a colorectal carcinoma and liver metastases. To reduce side-effects, patients were treated either with paracetamol or indomethacin. Higher MTDs were observed in patients treated with indomethacin. No detectable plasma TNF-α levels or TNF antibodies were measured under therapy (plasma TNF-α<20 pg/ml). We conclude that TNF-α appears to have some antineoplastic activity in patients with adenocarcinomas since 4 patients remained in no change or showed a minor response.

MRI findings in chronic hepatic encephalopathy depend on portosystemic shunt: results of a controlled prospective clinical investigation.

BACKGROUND/AIMS Deterioration of hepatic encephalopathy is a major concern with the transjugular intrahepatic portosystemic shunt procedure. Symmetric hyperintense globus pallidus on T1-weighted cranial magnetic resonance imaging in patients with liver cirrhosis anticipates hepatocerebral disease. It is hypothesized that hepatic encephalopathy and basal ganglia signal intensity progress in patients with cirrhosis of the liver undergoing transjugular intrahepatic portosystemic shunt. METHODS Twenty-four patients were randomized to undergo either transjugular intrahepatic portosystemic shunt or elective sclerotherapy. At study entry and 6 months after randomization, neurologic assessment, psychometric tests, standard EEG, and magnetic resonance imaging were performed. The severity of liver failure was graded using Child-Pughs classification. The signal intensity of the globus pallidus was determined on sagittal T1-weighted magnetic resonance imaging. RESULTS The T1-weighted signal intensity of the globus pallidus on magnetic resonance imaging significantly increased after transjugular intrahepatic portosystemic shunt placement (p<0.01), but not with elective sclerotherapy. At follow-up, neurological symptoms indicating decline of mental status and motor performance were somewhat more prevalent in transjugular intrahepatic portosystemic shunt patients. Significant deterioration of EEG abnormalities occurred in patients treated with transjugular intrahepatic portosystemic shunt as opposed to elective sclerotherapy (p<0.01). CONCLUSIONS Transjugular intrahepatic portosystemic shunt procedure increases hyperresonant globus pallidus on magnetic resonance imaging. Neuropsychiatric evaluation shows advancing hepatic encephalopathy, in particular with transjugular intrahepatic portosystemic shunt; however, it does not parallel the augmentation of pallidal signal intensity on magnetic resonance imaging.

Biliary secretion of bile acids and lipids in primary sclerosing cholangitis.Influence of cholestasis and effect of ursodeoxycholic acid treatment

In patients with primary sclerosing cholangitis cholestasis is a prominent feature of the disease. We studied the effect of cholestasis and of ursodeoxycholic acid treatment on the biliary secretion of bile acids and lipids in ten patients with primary sclerosing cholangitis. Ursodeoxycholic acid treatment for 3 months led to an increase in the biliary secretion rates of total bile acids from 0.91 mmol/h to 1.47 mmol/h, mainly due to an increase in urosodeoxycholic acid, which represented 31% of biliary bile acids. With increasing cholestasis, the biliary enrichment of the bile acid pool with urosodeoxycholic acid decreased. Biliary output of endogenous bile acids on average was unchanged, but in patients with cholestasis and diminished output before treatment, it increased after ursodeoxycholic acid. Phospholipid secretion increased from 0.26 mmol/h to 0.43 mmol/h without correlation to the degree of cholestasis. Biliary cholesterol secretion on average was unchanged after ursodeoxycholic acid (0.1 versus 0.09 mmol/h) but, in patients with cholestasis and diminished output before treatment, it increased after ursodeoxycholic acid. The decreasing enrichment of the bile acid pool with ursodeoxycholic acid with increasing cholestasis may be related to its slight effect in advanced disease. The increase in biliary phospholipid secretion may represent another mechanism of action of urosodeoxycholic acid responsible for its beneficial effect in cholestatic liver disease.

Selective inhibition of long-chain fatty acid uptake in short-term cultured rat hepatocytes by an antibody to the rat liver plasma membrane fatty acid-binding protein.

Uptake of long-chain fatty acids by short-term cultured hepatocytes was studied. Rat hepatocytes, which were cultured for 16 h on plastic dishes (3.6 X 10(6) cells/dish), were incubated with [3H]oleate in the presence of various concentrations of bovine serum albumin as a function of the concentration of unbound [3H]oleate in the medium. At 37 degrees C initial uptake velocity (V0) was saturable (Km = 9 X 10(-8) M; Vmax = 835 pmol/min per mg protein). V0 was temperature dependent with an optimum at 37 degrees C and markedly reduced at 4 degrees C and 70 degrees C. To evaluate the biologic significance of a previously isolated rat liver plasma membrane fatty acid-binding protein as putative carrier protein in the hepatocellular uptake of fatty acids, cultured hepatocytes were treated with a monospecific rabbit antibody (IgG-fraction) to this membrane protein or the IgG-fraction of the pre-immune serum as controls. Uptake kinetics of [3H]oleate in antibody pretreated short-term cultured hepatocytes revealed a depression of Vmax by 70%, while Km was only reduced by 16% compared to controls, indicating a predominant non-competitive type of inhibition. V0 of a variety of long-chain fatty acids (oleic acid, arachidonic acid, palmitic acid, stearic acid) was reduced by 56-69%, while V0 of [35S]sulfobromophthalein, [3H]cholic acid and [14C]taurocholic acid remained unaltered. These data support the concept that in the system of cultured hepatocytes, uptake of long-chain fatty acids is mediated by the rat liver plasma membrane fatty acid-binding protein.

Synthesis of the X-protein of hepatitis B virus in vitro and detection of anti-X antibodies in human sera.

A protein of 154 amino acids, predicted to be encoded by the X-open reading frame of the hepatitis B virus (HBV) genome, was synthesized in an in vitro translation system from SP6 transcripts containing the X-coding sequence. As characterized by SDS-PAGE and immunoprecipitation this X-protein possesses the expected molecular weight of 17 kDa and reacts specifically with rabbit antisera directed against a fusion protein from Escherichia coli that contained 145 of the 154 amino acids from the X-sequence. The X-protein, radiolabeled with [35S]methionine, provided a sensitive and specific antigen to screen for anti-X antibodies in sera from HBV patients. Positive signals were obtained preferentially in subjects suffering from HBV-induced liver cirrhosis or primary hepatocellular carcinoma (PHC), i.e., individuals that had been exposed for an extended time period to HBV gene products. Carefully controlled experiments failed to reveal the presence of X-related proteins specific to liver specimens from HBV patients.