sn-1,2-Diacylglycerols and phorbol diesters stimulate thromboxane synthesis by de novo synthesis of prostaglandin H synthase in human promyelocytic leukemia cells.

We studied the regulation of thromboxane (TX) synthesis in promyelocytic leukemia cells during macrophage differentiation. Cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) showed rates of TXB2 synthesis from exogenous arachidonic acid that exceeded that of control cells by a factor of up to 81. Cells treated with sn-1,2-dioctanoylglycerol (diC8) showed similarly high TXB2 synthesis rates when diC8 was added concomitantly with a subthreshold concentration of TPA or when given in multiple doses. These activities depended on de novo synthesis of prostaglandin H (PGH) synthase because: microsomal PGH synthase activity showed large increases in Vmax values, and mass measurements of PGH synthase revealed the presence of PGH synthase in differentiating cells whereas the enzyme was undetectable in control cells. These results indicate that macrophage differentiation is associated with stimulation of TXB2 synthesis that requires both activation of protein kinase C and de novo synthesis of PGH synthase.

Human platelet-derived growth factor stimulates prostaglandin synthesis by activation and by rapid de novo synthesis of cyclooxygenase.

Human platelet-derived growth factor (PDGF) stimulated prostaglandin (PG) E2 synthesis in the cell cycle of Swiss 3T3 cells at two distinct time intervals, with a first plateau within 10 min and a second plateau within 2-4 h after addition of PDGF. At 4 h, the concentration of PGE2 in PDGF-stimulated cultures exceeded the quiescent control cells by a factor of 10-15. Quiescent cells incubated with up to 16 microM exogenous arachidonic acid (AA) synthesized only small amounts of PGE2. In contrast, 4 h after addition of PDGF, the concentration of PGE2 synthesized from exogenous AA exceeded that in quiescent cultures by a factor of 28. The effect of PDGF stimulation on PG synthesis from exogenous AA could not be explained by growth factor-mediated increase in the cellular free AA pool as shown in experiments using [14C]AA. PDGF also stimulated synthesis of PGI2 (prostacyclin), thromboxane, and PGF2 alpha from exogenous AA. While inhibition of protein synthesis by 10 micrograms/ml cycloheximide had no effect on the early increase in PGE2 synthesis, the second increase was completely prevented. Additionally, cycloheximide treatment at 6 h after PDGF stimulation resulted in rapid decline of PGE2 synthesis from exogenous AA. Quiescent cultures pretreated with 100 microM aspirin and stimulated by PDGF thereafter recovered from cyclooxygenase inhibition within 180 min. Our results suggest that phospholipase activation and resultant AA release is not sufficient to induce the burst of PG synthesis observed in PDGF-stimulated cells. Instead, PDGF stimulates PG synthesis by direct effects on the PG-synthesizing enzyme system, one involving a protein synthesis-independent mechanism and another that requires rapid translation of cyclooxygenase.

Chronic ursodeoxycholic acid- and chenodeoxycholic acid-feeding-induced changes of colon mucosal cell proliferation in rats.

Hyperproliferation has been suggested to play a major role in bile acid-dependent colorectal tumor promotion. Effects of chronic feeding of chenodeoxycholic acid (CDC) and ursodeoxycholic acid (UDC) were tested on cell proliferation in the colon of male noninbred Wistar rats. By use of a dynamic method measuring actual rates of cell production, proliferation was modulated by both bile acids only in the proximal part of the colon. UDC feeding produced mild hyperproliferation of basal crypt cells (cell position 5-8: 7.6 +/- 2.0 vs. 3.5 +/- 1.3 cells/1,000 cells/hr--P less than .05; cell position 9-12: 18.1 +/- 10.7 vs. 10.3 +/- 2.9--P less than .05; cell position 13-16: 18.1 +/- 8.9 vs. 9.1 +/- 2.3--P less than .05). This finding reflected a characteristic compensatory response to superficial cell damage. However, CDC application did not effect cell regeneration in this crypt area but led to a striking drop of cell renewal in higher crypt cell positions (positions greater than or equal to 17), where no proliferation was detectable. These data suggest that CDC exerts its tumor-promoting effect by other means than hyperproliferation.

Induction and activation of rat liver microsomal bile salt glucuronyltransferase.

In vivo induction and in vitro activation of the recently described bile salt glucuronyltransferase were investigated in rat. A radioactive assay for the determination of glucuronyltransferase activity was used. 14C-Labeled bile salts served as substrates, and the glucuronides were separated by thin layer chromatography. Lithocholate glucuronyltransferase activity was determined in liver microsomes of phenobarbital- and 3-methylcholanthrene-treated rats and of untreated controls. Pretreatment with phenobarbital induced lithocholate glucuronyltransferase activity to 150.5% of controls. In contrast, 3-methylcholanthrene treatment decreased activity to 29.6% of controls. In vivo activation of lithocholate glucuronyltransferase by Triton X-100 was observed in controls and in the 3-methylcholanthrene group, but not in the phenobarbital group. Substrate activation of the enzyme by lithocholate was demonstrated in microsomes of untreated controls. Pretreatment with 3-methylcholanthrene, but not phenobarbital, increased the latency of lithocholate glucuronyltransferase. The results indicate that rat liver microsomal bile salt glucuronyltransferase activity is increased by in vivo induction with phenobarbital and by in vitro activation with detergents like Triton X-100. The induction of bile salt glucuronide formation by phenobarbital is most likely one of the factors contributing to the increased biliary and fecal excretion of bile salts in patients with cholestasis following phenobarbital therapy.

Taurine and glycine conjugation and sulfation of lithocholate in primary hepatocyte cultures.

Rat primary liver cells were used to study taurine and glycine conjugation and sulfation of lithocholate. After addition of [14C]lithocholate to the tissue culture medium, synthesis and excretion of amidated and/or sulfated products were investigated for up to 24 h. After incubation for 1 h, more than 83% of the labeled bile salt was amidated but not sulfated and between 5 and 11% was sulfated, with more than 80% of the sulfated bile salts being also amidated. After 24 h, the proportion of sulfated lithocholate had increased to about 23% and more than 99% of the lithocholate sulfate was additionally conjugated with glycine or taurine. Both sulfates and non-sulfates were preferably amidated with taurine. We conclude that in primary rat hepatocytes, (1) lithocholate is rapidly and almost completely conjugated with glycine or taurine (amidated), whereas sulfation of lithocholate (and its amidates) proceeds slowly and even after 24 h represents only a small proportion of the total lithocholate metabolites, and (2) sulfated and unsulfated bile salts are both preferably amidated with taurine.

Hepatitis C virus, alcoholic cirrhosis, and hepatocellular carcinoma.

We undertook a retrospective study to determine the prevalence of hepatitis C virus (HCV) and hepatitis B virus (HBV) infection in 81 Caucasian patients with confirmed hepatocellular carcinoma (HCC). Besides HBV and HCV serological markers, HCV RNA and HBV DNA were detected in serum and liver tissue by polymerase chain reaction. Overall, HCV RNA was found in 20 cases (25%), HBV DNA in 21 patients (26%), and coinfection in 3 patients (3%). HCV RNA in liver tissue was not found without virus in serum, whereas HBV DNA was found in the liver tissue of one patient without viremia. In an additional analysis, 32 patients with HCC and alcoholic cirrhosis (HCC-AC) were compared to 35 cases with AC without HCC and 35 cases with alcoholic hepatitis. The prevalence of HCV RNA in HCC-AC (19%) was significantly higher than in the other groups (AC, 3%; alcoholic hepatitis, 0%). HBV DNA was present in 19% of HCC-AC as compared to 3% of AC and 0% of alcoholic hepatitis. We conclude that the form of HCC in 50% of the patients in a Western European country is related to chronic viral hepatitis. Our data obtained from a group of patients having alcoholic liver disease with or without HCC suggest that the prevalence of HCV RNA or HBV DNA in these populations increases with the severity of hepatic injury.

Effect of intraperitoneal recombinant human tumour necrosis factor alpha on malignant ascites.

29 patients with refractory malignant ascites due to metastatic peritoneal spread of adenocarcinomas originating from the ovary, gastrointestinal tract, liver, breast and uterus were treated in a phase I trial of intraperitoneal infusions of recombinant human tumour necrosis factor alpha (rhTNF-alpha). Patients received 40-350 micrograms/m2 rhTNF-alpha intraperitoneally once weekly for 2 months or for a shorter period in case of early resolution of ascites. Systemic side-effects resembled those reported for rhTNF-alpha given intravenously. No dose-limiting toxicities were found and thus a maximum tolerated dose of intraperitoneal rhTNF-alpha was not established. Out of 29 patients, 22 responded with a complete (16) or partial (6) resolution of their ascites. There was a less than 50% reduction in 4, and no increase in ascites in 1. 1 patient showed progressive ascites formation, and another patient was not eligible because of early death unrelated to treatment. Trials in patients with smaller tumour burden are warranted.

Prostanoid release in experimental liver transplantation.

Prostanoids are biologically active mediators of inflammation and tissue injury. To investigate the role of prostanoids in orthotopic liver transplantation we used a porcine model and determined prostaglandin E2, 6-keto-prostaglandin F1 alpha, and thromboxane B2 in arterial, portal, and hepatic venous blood during organ harvesting, the recipient operation, and the early postoperative period. There were no significantly increased serum levels during the donor operation or at the end of cold storage. As early as 1 or 5 min after initiation of reperfusion of the transplanted organ, prostanoids in hepatovenous blood increased dramatically (100-500-fold). Changes in arterial and portal blood (10-50-fold) were less pronounced but still statistically significant. In surviving animals these values returned to normal within 24 hr. The hepatic release of metabolites of the arachidonic acid cascade after liver grafting indicates that the synthesis of prostanoids might contribute to morphological and functional alterations of the transplanted graft. In addition, the increased arterial values of circulating prostanoids may potentially participate in severe cardiovascular, hemostatic, and immunological alterations known to occur after liver transplantation.

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

Fifteen patients with advanced metastatic adenocarcinomas were treated in a phase-I study with continuous intravenous 24 h infusion of recombinant tumor necrosis factor alpha (TNF-alpha) in order to determine the maximum tolerated dose (MTD) and associated side-effects. Patients received 40-400 micrograms/m2 TNF-alpha 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 micrograms/m2 for 24 h, were fever, chills, fatigue, 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-alpha levels or TNF antibodies were measured under therapy (plasma TNF-alpha less than 20 pg/ml). We conclude that TNF-alpha appears to have some antineoplastic activity in patients with adenocarcinomas since 4 patients remained in no change or showed a minor response.

Synaptophysin identified in metastases of neuroendocrine tumors by immunocytochemistry and immunoblotting.

Synaptophysin, an Mr 38,000 integral membrane glycoprotein of neurotransmitter vesicles, has been identified in diverse primary neuroendocrine (NE) tumors of both neural and epithelial origin (Wiedenmann and co-workers, Proc Natl Acad Sci USA 1986; 83: 3500-3504). In the present study, metastases of several types of NE tumors, including medullary thyroid carcinoma, gastrinoma, insulinoma, small (oat) cell carcinoma of the lung, gastrointestinal carcinoid, and neuroblastoma, were examined for the presence of synaptophysin by immunocytochemistry, with the use of tissue sections as well as centrifuged cell suspensions and by immunoblotting of tumor proteins. The results show that expression of synaptophysin can be maintained during formation of metastases. Therefore, the authors propose that synaptophysin antibodies be used for the positive identification of metastatic NE tumors, notably in differential diagnosis. The possible implications of these findings for tumor diagnosis are discussed.

Follow-up of patients with hepatitis non-A, non-B: incidence and persistence of anti-HCV depend on route of transmission.

Of 32 patients with non-A, non-B hepatitis, 10 (31%) were still anti-HCV-positive 12.8 years after the acute phase of the disease. Seven of the patients (21.9%) still had elevated ALT levels, and among these, 5 out of 5 patients who had been subject to parenteral risk were anti-HCV-positive. In contrast, none of the patients who had not been subject to parenteral risks were positive.

Beneficial effect of somatostatin on galactosamine induced liver injury.

The purpose of our investigation was to study the effect of somatostatin on acute experimental liver injury induced in rats by galactosamine (1.2 g/100 g body wt.). Somatostatin (125 micrograms/100 g body wt.) was administered subcutaneously in a protamine sulphate/ZnCl2 suspension either 2 h prior to the injection of galactosamine or 2 h and again 12 h following the injection. Serum transaminases (GOT, GPT) and serum concentrations of triiodothyronine and thyroxine were determined 28 h after the injection of galactosamine. Histology of the liver was performed by light microscopy. Our results showed that the administration of somatostatin significantly (P less than 0.02) reduced the elevation of GOT and GPT activity and diminished the degree of necrosis, and that although the administration of dibutyryl-cAMP (5 mg/100 g body wt.) intensified galactosamine induced liver injury, this effect of dibutyryl-cAMP could be completely prevented by somatostatin treatment. There was no difference in the serum concentrations of triiodothyronine and thyroxine in controls as compared to galactosamine and galactosamine plus somatostatin treated rats. At present the mechanism of this cytoprotection by somatostatin is unknown.

Microsomal ethanol oxidation in the colonic mucosa of the rat. Effect of chronic ethanol ingestion.

A microsomal ethanol oxidizing system (MEOS) is present in the colonic mucosa of the rat. This MEOS metabolizes ethanol to acetaldehyde at the physiological pH of 7.4. Alcohol dehydrogenase or catalase are not involved in the reaction. The Michaelis Menten constant of the reaction is 13.7 +/- 0.3 mM and the maximal velocity is 219 +/- 30 pmoles acetaldehyde/mg microsomal protein X min. Bacterial ethanol metabolism does not contribute to the acetaldehyde production in the colonic MEOS. Chronic ethanol consumption has no effect on colonic MEOS activity. In addition, chronic ethanol ingestion does not affect colonic microsomal NADPH-cytochrome-c-reductase nor benzo(a) pyrene hydroxylase activity.

A new radioimmunoassay for demonstrating the antibody to hepatitis-B-surface-antigen(HBs-Ag).

A new radioimmunological system for demonstrating the antibody to hepatitis-B-surface-antigen (HBS-Ag) based on the immunoadsorption principle is described. The optimal conditions for the test are given. False-negative and false-positive results could be excluded by specificity tests on sera from 100 voluntary blood donors. The sensitivity of the system is comparable to that of other more expensive radioimmunological methods.

Gilbert's syndrome: diagnosis by typical serum bilirubin pattern.

Analysis of serum unconjugated and conjugated bilirubin fractions by routine diazo procedures does not allow a definite diagnosis of Gilbert's syndrome. By the alkaline methanolysis procedure of Blanckaert followed by thin-layer chromatography we were able to discriminate Gilbert's syndrome even in the presence of normal serum bilirubin concentrations from healthy subjects, patients with chronic persistant hepatitis and patients with chronic hemolysis. The relative proportion of unconjugated bilirubin in serum was 95 +/- 2% in patients with Gilbert's syndrome (n = 28), 84 +/- 5% in healthy subjects (n = 29), 75 +/- 6% in patients with chronic persistant hepatitis (n = 7) and 85 +/- 3% in patients with chronic hemolysis (n = 9). The difference between Gilbert's syndrome and the control groups with normal or elevated serum bilirubin was highly significant (p less than 0.001). In Gilbert's syndrome, unconjugated bilirubin ranged between 90 and 99%, in healthy subjects between 72 and 90%, in patients with chronic persistant hepatitis between 68 and 85% and in patients with chronic hemolysis between 81 and 89% of total. An overlap was only seen in one patient with Gilbert's syndrome and in 2 healthy subjects at the 90% level. We conclude that in most patients with Gilbert's syndrome provocation tests are no longer necessary.

Biliary excretion of iron in healthy man and in patients with alcoholic cirrhosis of the liver.

We measured the biliary excretion of iron in 11 patients with alcoholic cirrhosis of the liver and in 10 healthy controls using an intestinal perfusion technique. In the patients with cirrhosis increased amounts of iron in liver tissue were present. The concentrations of iron in the bile samples were determined by atomic absorption spectrometry. The biliary excretion of iron in the healthy controls was 0.32 +/- 0.09 mumol/h and in the patients with cirrhosis it was 0.45 +/- 0.14 mumol/h. The biliary excretion of iron in the patients with cirrhosis was not reduced, indicating that other mechanisms than a reduced biliary excretion of iron must be responsible for the accumulation of iron in liver tissue in alcoholic cirrhosis.

Colonic cyclic AMP metabolism following chronic ethanol consumption in the rat: effect of hormonal secretagogues.

The colonic cyclic AMP system is known to be involved in intestinal secretion and can be stimulated by a variety of gastrointestinal hormones including prostaglandins. We have investigated the effect of chronic ethanol ingestion on the activity of the key enzymes in cyclic AMP metabolism--adenylate cyclase and cyclic AMP phosphodiesterase--in the colonic mucosa of the rat. Chronic ethanol consumption by feeding a nutritionally adequate liquid diet enhanced basal colonic adenylate cyclase activity significantly by 168% (p less than 0.01), but had no effect on colonic low Km cyclic AMP phosphodiesterase activity. In addition, various hormonal secretagogues were used to stimulate colonic adenylate cyclase. Colonic adenylate cyclase exhibited a significantly greater sensitivity and efficacy to prostaglandins and vasoactive intestinal peptide after chronic ethanol ingestion. Since increased intestinal cyclic AMP production due to an increased activity of intestinal adenylate cyclase is known to promote intestinal secretion of water and electrolytes, the frequently observed diarrhea in alcoholics may be explained at least in part by an enhanced production of colonic cyclic AMP.

Intramural hematoma of the esophagus: a complication of carbon tetrachloride intoxication with acute renal failure.

Following a period of prolonged severe vomiting, an intramural esophageal hematoma could be demonstrated by endoscopy and by X-ray in a 21 year old patient with hepatic and renal failure after exposure to carbon tetrachloride. The hematoma resolved spontaneously. The intramural hematoma is thought to have developed from a microdissection of the esophageal wall in the presence of a hemorrhagic diathesis.

Ethanol and intestinal carcinogenesis in the rat.

The effect of chronic ethanol administration on 1,2-dimethylhydrazine induced rectal carcinogenesis was investigated in 32 paired male Sprague-Dawley rats fed a nutritionally adequate liquid diet containing 36% of total calories either as ethanol or isocaloric carbohydrates. Chronic ethanol ingestion increased the total number of rectal tumors significantly (17 vs. 6, p less than 0.02), whereas no cocarcinogenic effect of ethanol was observed in other parts of the intestine. Alcohol did not influence tumor size or histopathology. A 47% increase in the activity of mucosal alcohol dehydrogenase in the distal colorectum was found between chronically ethanol fed and pair fed controls (0.241 +/- 0.019 vs. 0.164 +/- 0.020 mumol mg protein-1 hr-1, p less than 0.01). This could in part explain the cocarcinogenic effect of alcohol in this tissue. The data give experimental support to the epidemiologic findings of an increased incidence of rectal cancer in the alcoholic.

Detection of antibodies to proteins encoded by the X-region of hepatitis B virus (HBV) in sera of patients with chronic HBV infection: correlation with other HBV markers.

The genome of hepatitis B virus (HBV) contains a fourth open reading frame (ORF), designated X-region. It was the aim of our study to test sera of patients with chronic HBV infection for the presence of antibodies reactive with a 17 kd gene product of the X-ORF. For this purpose, a 35S-X-ORF-encoded protein, synthesized in vitro, was applied as antigen for the detection of antibodies to HBx proteins in sera of 86 individuals. Antibodies reacting with a gene product of the X-ORF were present in 10 out of 24 HBsAg-positive patients with hepatocellular carcinoma (PLC) or liver cirrhosis and in one out of 8 HBsAg-carriers. In addition, the antibodies could also be detected in 6 out of 35 sera from patients with PLC or cirrhosis negative for HBsAg but positive for anti-HBc and anti-HBs. Antibodies to a gene product of the X-ORF can be detected in sera of patients with chronic HBV-related liver disease, independently of HBsAg and the HBeAg/anti-HBe system.