Generation of a novel antibody probe to the apical sodium-dependent bile acid transporter that inhibits ileal bile acid absorption.

Intestinal bile acid absorption is mediated by a sodium-dependent transporter located in the brush border apical membrane of ileocytes. The transmembrane topology and the role of individual amino acid residues in the bile acid transport process have been investigated by means of various experimental approaches, leading to multiple hypotheses. We raised a monoclonal antibody against a segment of the transporter comprising vicinal cysteine residues, in order to evaluate its functional role. A 14 amino acid peptide, corresponding to amino acids 104-117 of the transporter, was synthesized, and a monoclonal anti-peptide antibody was raised. In vitro uptake-inhibition studies in the presence of the monoclonal anti-peptide antibody were performed using ileal brush border membrane vesicles. Rabbit ileum was perfused in vivo with 5 mM taurocholic acid in the presence of the monoclonal antibody, and bile acid absorption inhibition was evaluated. The anti-peptide monoclonal antibody significantly reduced the in vitro uptake and in vivo absorption of taurocholic acid. The present data demonstrate the functional relevance of the 104-117 peptide segment and report the generation of a novel antibody against the apical sodium-dependent bile acid transporter (ASBT) that may be used as a therapeutic agent in hypercholesterolemia and in cholestatic pruritus.

Hepatobiliary phospholipid transporter ABCB4, MDR3 gene variants in a large cohort of Italian women with intrahepatic cholestasis of pregnancy.

BACKGROUND: Intrahepatic cholestasis of pregnancy is a multifactorial disorder of pregnancy associated with a genetic background. AIM: To evaluate the genetic contribution of ABCB4, MDR3 gene in the development of intrahepatic cholestasis of pregnancy in a large cohort of Italian subjects. METHODS: This study represents an extension of a previous multicentre-prospective study including three Italian referral centres. In all, we enrolled 96 women at the 3rd trimester of pregnancy. Genomic DNA was extracted from peripheral venous blood leucocytes by standard procedures. Polymerase chain reaction was used to amplify exon 14, 15 and 16 of MDR3 gene. RESULTS: We found 3 non-synonymous heterozygous mutations in exon 14 (E528D, R549H, G536A), 1 in exon 15 (R590Q) and 2 in exon 16 (R652G, T6671). MDR3 gene variants in exons 14, 15 and 16 occurred in 7/96 of pregnant mothers with intrahepatic cholestasis of pregnancy (7.2%), and in none of 96 pregnant controls matched for age and parity. All seven patients had normal gamma-glutamyl transpeptidase, normal bilirubin, but high levels of both alanine transferase and serum bile acids. One had cholesterol biliary lithiasis. The outcome of pregnancy was normal in four cases (with vaginal delivery), while there was one fetal distress. CONCLUSIONS: MDR3 mutations are responsible for the development of intrahepatic cholestasis of pregnancy in only a small percentage of Italian women. Further genetic studies are warranted, however, to clarify the role of different mutations in intrahepatic cholestasis of pregnancy.

Transjugular intrahepatic portosystemic shunt: a case report of rescue management of unrestrainable variceal bleeding in a pregnant woman.

Liver cirrhosis complications in pregnant women are frequent and death rate secondary to variceal bleeding is relevant. Both sclerotherapy and banding ligation seem to be safe procedures in pregnancy; when bleeding is not arrested endoscopically an emergency transjugular intrahepatic portosystemic shunt should be considered, but data regarding pregnant cirrhotic women are scarce. We describe the case of a pregnant woman at 14 weeks of gestation who underwent management of acute variceal bleeding by transjugular intrahepatic portosystemic shunt. Transjugular intrahepatic portosystemic shunt may represent a rescue treatment for failed attempts of band ligation or sclerotherapy.

Clinical trial: modulation of human placental multidrug resistance proteins in cholestasis of pregnancy by ursodeoxycholic acid.

BACKGROUND: The effects of ursodeoxycholic acid on human placental bile acids and bilirubin transporters in intrahepatic cholestasis of pregnancy are still undefined. AIM: To evaluate whether ursodeoxycholic acid affects MRP2, MRP3 and MRP4 expression in the placenta. MATERIALS AND METHODS: Forty-three pregnant women were enrolled; fourteen subjects had physiological pregnancies. Intrahepatic cholestasis of pregnancy patients were divided into two groups: (i) 13 received ursodeoxycholic acid (20 mg/kg/day) and (ii) 16 untreated. Total bile acid and bilirubin in serum and cord blood were determined in each subject. Multidrug resistance proteins expression (immunoblot, quantitative real-time PCR) was evaluated in placentas collected at delivery. anova test was used for statistical analysis of data. RESULTS: Ursodeoxycholic acid administration significantly improved maternal serum bile acid and cord blood bilirubin and bile acid levels. MRP2 protein and RNA expression was significantly increased in placentas from treated patients compared to controls (P < 0.001 and P < 0.01, respectively). MRP3 protein expression was not significantly different between the groups while RNA expression was significantly decreased in treated patients (P < 0.01). MRP4 did not show significant differences between the groups. CONCLUSIONS: Ursodeoxycholic acid administration induces placental MRP2 expression, and reduces bilirubin and bile acid levels in cord blood.

Ursodeoxycholic acid improves gastrointestinal motility defects in gallstone patients.

AIM: To simultaneously evaluate the presence of defects in gallbladder and gastric emptying, as well as in intestinal transit in gallstone patients (GS) and the effect of chronic ursodeoxycholic acid (UDCA) administration on these parameters and on serum bile acids and clinical outcome in GS and controls (CTR). METHODS: After a standard liquid test meal, gallbla-dder and gastric emptying (by ultrasound), oroileal transit time (OITT) (by an immunoenzymatic technique) and serum bile acids (by HPLC) were evaluated before and after 3 mo of UDCA (12 mg/kg bw/d) or placebo administration in 10 symptomatic GS and 10 matched healthy CTR. RESULTS: OITT was longer in GS than in CTR (P < 0.0001); UDCA significantly reduced OITT in GS (P < 0.0001), but not in CTR. GS had longer gastric half-emptying time (t(1/2)) than CTR (P < 0.0044) at baseline; after UDCA, t(1/2) significantly decreased (P < 0.006) in GS but not in CTR. Placebo administration had no effect on gastric emptying and intestinal transit in both GS and CTR. CONCLUSION: The gallstone patient has simultaneous multiple impairments of gallbladder and gastric emptying, as well as of intestinal transit. UDCA administration restores these defects in GS, without any effect in CTR. These results confirm the pathogenetic role of gastrointestinal motility in gallstone disease and suggest an additional mechanism of action for UDCA in reducing bile cholesterol supersaturation.

A statistical model predicting high hepatocyte proliferation index and the risk of developing hepatocellular carcinoma in patients with hepatitis C virus-related cirrhosis.

BACKGROUND: Incidence of hepatocellular carcinoma in hepatitis C virus-related cirrhosis is 4% per year. Although cost-effective, current screening could be improved. AIM: To develop a statistical model including non-invasive parameters able to identify patients at high risk of developing hepatocellular carcinoma. METHODS: One hundred and fifty-eight patients (73F:85M) with compensated chronic hepatitis C virus liver disease underwent evaluation, including argyrophilic nucleolar organizer regions proliferation index, and were followed up for 56.18 +/- 1.44 months. RESULTS: Fifty-six patients had chronic hepatitis without cirrhosis and low argyrophilic nucleolar organizer regions proliferation index (< or =25%), 65 had hepatitis C virus-related cirrhosis and low argyrophilic nucleolar organizer regions proliferation index and 37 had hepatitis C virus-related cirrhosis and high argyrophilic nucleolar organizer regions proliferation index (>25%). Groups were similar for gender and viral genotype distribution. None of the patients with chronic hepatitis without cirrhosis developed hepatocellular carcinoma, compared with 6.1% of low argyrophilic nucleolar organizer regions proliferation index and 30.6% of high argyrophilic nucleolar organizer regions proliferation index (P = 0.002). By multivariable logistic regression analysis, the following parameters were independently associated with hepatocellular carcinoma development and used for the development of the statistical model: platelets (OR 0.98), gamma-globulins (OR 0.111), alanine aminotransferase/aspartate aminotransferase ratio (OR 0.07), serum ferritin (OR 1.0) and ultrasonographic pattern (coarse OR 2.9, coarse nodular OR 10.12). The statistical model properly allocated 95.9% of patients with low argyrophilic nucleolar organizer regions proliferation index and 72.2% of patients with high argyrophilic nucleolar organizer regions proliferation index. CONCLUSIONS: The model, to be validated in large prospective studies, may help tailoring screening according to the risk of hepatocellular carcinoma development.

Intrahepatic cholestasis of pregnancy: three novel MDR3 gene mutations.

BACKGROUND: The aetiology of intrahepatic cholestasis of pregnancy is unknown, but more than 10 different MDR3 gene mutations have recently been identified. AIM: To evaluate the genetic contribution of the MDR3 gene in the pathogenesis of intrahepatic cholestasis of pregnancy in Italian subjects. METHODS: We performed a multicentre prospective case-control study, enrolling 80 women with intrahepatic cholestasis of pregnancy at the third trimester of pregnancy and 80 pregnant women without intrahepatic cholestasis of pregnancy. Genomic DNA was extracted from peripheral venous blood leucocytes using standard procedures. The polymerase chain reaction was used to amplify exon 14 of the MDR3 gene and the polymerase chain reaction products were sequenced using a Big Dye Terminator Cycle Sequencing kit. RESULTS: Three novel non-synonymous heterozygous mutations in exon 14 were found (4%; E528D, R549H, G536R) among the 80 intrahepatic cholestasis of pregnancy patients, whereas the pregnant controls were all negative for exon 14 polymorphisms. The three patients involved had normal GGT and bilirubin, but high levels of both ALT and serum bile acids. One had cholesterol bile stones. The outcome of pregnancy was normal for two (with vaginal delivery), while foetal distress was recorded in the third. CONCLUSIONS: These three novel mutations add further information on the involvement of the MDR3 gene in intrahepatic cholestasis of pregnancy. As in other studies, we found only heterozygous mutations that could cause an impaired transport protein function, not its absence (which is responsible for more severe liver disease). Different genetic backgrounds might justify the presence of novel MDR3 gene mutations.

Higher doses of peginterferon alpha-2b administered twice weekly improve sustained virological response in difficult-to-treat patients with chronic hepatitis C: results of a pilot randomized study.

SUMMARY: Beside substantial progress in treatment of chronic hepatitis C (CHC) particular patients (genotype 1/4, high viral load, previous nonresponse, cirrhosis) remain difficult to treat. The aim of our pilot randomized study was to compare efficacy and tolerability of standard doses of Peginterferon alpha-2b + ribavirin with higher doses of Peginterferon alpha-2b administered twice weekly + ribavirin. Sixty-five outpatients with CHC were subsequently enrolled. Group A (n = 22) received recommended doses of Peginterferon alpha-2b and group B (n = 43), received high doses twice weekly. Groups were comparable for baseline characteristics. All genotype 1/4 patients had high baseline viraemia. Sustained virological response (SVR) was significantly higher in group B among naïve patients (72%vs 25%, P = 0.024). A significantly higher rate of SVR was observed in group B both considering only genotype 1/4 patients, (46%vs 13%, P = 0.03) and grouping together genotype 1/4 naive and relapsers (57%vs 11%, P = 0.039). Discontinuation rate was 32% (7 of 22) in group A and 21% (9 [corrected] of 43) in group B. Our response rates are the highest reported for genotype 1/4 with high viraemia. Our pilot study supports the need of randomized studies to evaluate both viral kinetics and efficacy of high dose and twice weekly administration of Peginterferon alpha-2b in genotype 1/4 patients with high viraemia who may need personalized treatment schedules.

Ten year incidence of HCV infection in northern Italy and frequency of spontaneous viral clearance.

BACKGROUND: Little is known of the incidence of hepatitis C virus (HCV) infection, and the frequency of spontaneous viral clearance in the general population is unknown. We conducted an epidemiological study in two Apennine towns in northern Italy. METHODS: Anti-HCV (ELISA and RIBA third generation) and HCV-RNA by polymerase chain reaction were tested in thawed sera from an adult general population of Loiano-Monghidoro in 1986 and 1996, obtained in the context of the MICOL (Multicenter Italian Study on Cholelithiasis). In 1999, anti-HCV positive subjects and sex and age matched controls were recalled in order to identify risk factors for acquiring HCV infection and to assess the family composition of anti-HCV+ subjects. RESULTS: For 1646 subjects, sera were available from both 1986 and 1996 (mean age in 1986 43 (0.39) years). In 1986, 57 (3.46%) subjects were HCV antibody positive (HCV-Ab+). Eight new cases were recorded in 1996: adult incidence was 50.3 cases/100 000 inhabitants/year. Fifty three of 63 (84.1%) HCV-Ab+ sera were also HCV-RNA+. Genotype 2a/2c accounted for 44% and 1b for 47.0% of cases. HCV-Ab+ subjects had higher serum levels of alanine aminotransferase with respect to controls (p<0.005), as did subjects infected with genotype 1 with respect to those with genotype 2 (p<0.05). Eleven of 65 (16.9%) HCV-Ab+ subjects spontaneously cleared HCV-Ab; 7/11 also lost HCV-RNA- in both serum and leucocytes. Sixteen anti-HCV+ subjects belonged to families containing more than one infected member. Married couples accounted for 10 of these 16 subjects. In four of these five married couples, HCV genotype was identical in the two spouses. CONCLUSIONS: In rural northern Italy, the adult incidence of HCV is approximately 50 cases/100 000 inhabitants/year. Our findings suggest that as many as 17% of infected subjects may spontaneously clear HCV-Ab. Interfamilial transmission seems to have a role in the spread of infection.

Gallbladder cholesterol polyps and cholesterolosis.

Cholesterolosis of the gallbladder consists in an accumulation of cholesterol esters and triglycerides in the macrophages at gallbladder wall level and may be either diffuse or polypoid in form. A prevalence of 4-8% has been reported, particularly in the male sex; results concerning a relationship between cholesterolosis and lifestyle are controversial (alcohol intake, smoking habit), as well as the clinical and laboratory parameters, such as serum cholesterol and body mass index. Even more controversial is the relationship with gallstones which has been associated with the presence of cholesterol polyps only in a few surgical series. An increase in the activity of the cholesterol ester enzyme has been observed, in cholesterolosis patients, at gallbladder mucosa level which has led to the hypothesis of an increase in cholesterol ester deposit at this level; the hypothesis of an alteration in bile composition, in these patients, still remains to be elucidated. Ultrasonography is a sensitive tool in the diagnosis of cholesterolosis even if the use of echoendoscopy is becoming increasingly important in the differential diagnosis between benign and malignant polypoid lesions. Even if, in a few series, patients with polyps present a clinical pattern characterized by specific biliary symptoms, both in our experience and in that of others, symptoms are aspecific, the frequency of dyspeptic symptoms being comparable to that in the general population. The natural history of this lesion is, in general, benign and for polyps with size ranging from 6 mm to 10 mm a yearly follow-up with ultrasonography is advisable.

Improved liver tests and greater biliary enrichment with high dose ursodeoxycholic acid in early stage primary biliary cirrhosis.

BACKGROUND: Ursodeoxycholic acid is currently used for the treatment of primary biliary cirrhosis at 13-15 mg/kg/day, but liver tests of some patients do not return to normal at this dose. Studies reported here were designed to test whether a higher dose of ursodeoxycholic acid than is currently used would induce still greater biliary enrichment of ursodeoxycholic acid and whether such enrichment would lead to still further improvement in liver tests in patients with early primary biliary cirrhosis. METHODS: A total of 20 patients with histologically proven primary biliary cirrhosis were enrolled. Patients had early stage primary biliary cirrhosis as serum bilirubin levels were normal and the Mayo risk score 4.2 +/- 0.5. Group 1 received 600, 1200 and 1800 mg/day of ursodeoxycholic acid; group 2 received 900, 1500 and 2100 mg/day. The order of periods was randomized. Each treatment period lasted 3 months followed by a further 3 months during which a standard dose of ursodeoxycholic acid was given. At the end of each treatment period, liver tests were evaluated, and biliary bile acid pattern of duodenal bile was determined using high pressure liquid chromatography. RESULTS: Biliary bile acid became enriched in ursodeoxycholic acid in direct relationship to dosage [r = 0.84, p < 0.001). At doses of 1800 mg/day (25-35 mg/kg/day), biliary ursodeoxycholic acid averaged 69 +/- 6.6%. A progressive decrease of alanine aminotransferase [p < 0.0001), aspartate aminotransferase [p < 0.001) and alkaline phosphatase [p < 0.02) was observed with increasing concentrations of ursodeoxycholic acid in bile. Biochemical liver tests showed a stronger correlation with biliary concentrations of ursodeoxycholic acid than with the administered dose. CONCLUSIONS: In early primary biliary cirrhosis, higher dose ursodeoxycholic acid appears to be more effective than doses currently recommended.

[Cholic acid metabolism in human fecal cultures during diet supplementation with Lacto-bacillus rhamnosus GG]. / Metabolismo dell'acido colico in colture di feci umane in corso di assunzione di una dieta con aggiunta di Lactobacillus rhamnosus GG. Dati preliminari.

BACKGROUND: Lactobacillus rhamnosus GG is used as probiotic and is thought to have protective properties in the human gastrointestinal tract and in other organs. Enrichment of the bile acid pool with secondary bile acids is common in some hepatic and gastrointestinal diseases and this is considered as a pathogenic element in the disease progression. The aim of this study was to evaluate the effect of probiotic feeding on fecal bile acid biotransformation, particularly on the production of secondary bile acids. METHODS: Six normal volunteers were administered 500 g/die of a yogurt preparation containing 4 x 10(9) C.F.U. of Lactobacillus rhamnosus GG for 30 days. The 7alpha-dehydroxylation activity was investigated following addition of cholic acid to the stool samples collected before and after the probiotic feeding. The production of deoxycholic acid and the decrease in cholic acid level were studied. RESULTS: The comparison of biotransformation rate of cholic acid to deoxycholic acid before and after probiotic feeding didn't reach a statistically significant difference, but a strong difference was seen in three of the six subjects, indicating a different behavior in different groups of healthy subjects. Furthermore, the three non responder subjects had a lower fecal 7alpha-dehydroxylation activity in the stool samples from the pre-treatment collection. CONCLUSIONS: These results indicate that subjects with a higher production of secondary bile acids in stools may represent a target group for a larger trial with oral probiotic administration.

Adaptive regulation of bile salt transporters in kidney and liver in obstructive cholestasis in the rat.

BACKGROUND & AIMS: Cholestasis results in adaptive regulation of bile salt transport proteins in hepatocytes that may limit liver injury. However, it is not known if changes also occur in the expression of bile salt transporters that reside in extrahepatic tissues, particularly the kidney, which might facilitate bile salt excretion during obstructive cholestasis. METHODS: RNA and protein were isolated from liver and kidney 14 days after common bile duct ligation in rats and assessed by RNA protection assays, Western analysis, and tissue immunofluorescence. Sodium-dependent bile salt transport was also measured in brush border membrane vesicles from the kidney. RESULTS: After common bile duct ligation, serum bile salts initially rose and then declined to lower levels after 3 days. In contrast, urinary bile salt excretion rose progressively over the 2-week period. By that time, the ileal sodium-dependent bile salt transporter messenger RNA and protein expression in total liver had increased to 300% and 200% of controls, respectively, while falling to 46% and 37% of controls, respectively, in the kidney. Sodium-dependent uptake of (3)H-taurocholate in renal brush border membrane vesicles was decreased. In contrast, the multidrug resistance-associated protein 2 expression in the kidney was increased 2-fold, even 1 day after ligation. Immunofluorescent studies confirmed the changes in the expression of these transporters in liver and kidney. CONCLUSIONS: These studies show that the molecular expression of bile salt transporters in the kidney and cholangiocytes undergo adaptive regulation after common bile duct obstruction in the rat. These responses may facilitate extrahepatic pathways for bile salt excretion during cholestasis.

Antigen presentation by liver cells controls intrahepatic T cell trapping, whereas bone marrow-derived cells preferentially promote intrahepatic T cell apoptosis.

Systemic activation and proliferation of CD8(+) T cells result in T cell accumulation in the liver, associated with T cell apoptosis and liver injury. However, the role of Ag and APC in such accumulation is not clear. Bone marrow chimeras were constructed to allow Ag presentation in all tissues or alternatively to restrict presentation to either bone marrow-derived or non-bone marrow-derived cells. OVA-specific CD8(+) T cells were introduced by adoptive transfer and then activated using peptide, which resulted in clonal expansion followed by deletion. Ag presentation by liver non-bone marrow-derived cells was responsible for most of the accumulation of activated CD8(+) T cells. In contrast, Ag presentation by bone marrow-derived cells resulted in less accumulation of T cells in the liver, but a higher frequency of apoptotic cells within the intrahepatic T cell population. In unmodified TCR-transgenic mice, Ag-induced T cell deletion and intrahepatic accumulation of CD8(+) T cells result in hepatocyte damage, with the release of aminotransaminases. Our experiments show that such liver injury may occur in the absence of Ag presentation by the hepatocytes themselves, arguing for an indirect mechanism of liver damage.

Cellular localization and up-regulation of multidrug resistance-associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver.

The hepatic expression of the ATP-dependent conjugate export pump multidrug resistance-associated protein 2 (Mrp2) is diminished in experimentally induced models of cholestasis. In this study we have examined the localization and expression of Mrp3, another member of the multidrug resistance-associated protein family, in normal liver and after obstructive cholestasis in the rat. Indirect immunofluorescence and confocal microscopy were used to determine the tissue localization and Western blot analysis was performed to quantitate the expression. In normal rat liver Mrp3 was found on the basolateral membrane of cholangiocytes and a single layer of hepatocytes surrounding the central vein. Three and 7 days after bile duct ligation Mrp3 expression was significantly increased, predominantly in hepatocytes in the pericentral region. By 14 days all hepatocytes showed basolateral membrane labeling for Mrp3 at a time when apical Mrp2 staining was significantly diminished. Proliferating bile ducts continued to stain positive, although the intensity of staining did not seem to vary. After 14 days Western blot quantitation showed that Mrp3 had increased approximately 30-fold in total liver membranes. Quantitation of Mrp3 in membranes from isolated hepatocytes of livers of sham and common bile duct-ligated (CBDL) animals showed a significant up-regulation beginning at 1 day and continuing to increase through 14 days postligation. This was in contrast to the progressive decrease in Mrp2 protein. Because Mrp3 is capable of transporting toxic bile acids, up-regulation of Mrp3 may compensate for the down-regulation of Mrp2 in obstructive cholestasis.

Ursodeoxycholic acid administration in patients with cholestasis of pregnancy: effects on primary bile acids in babies and mothers.

Little is known about the effects on the fetus of ursodeoxycholic acid (UDCA) treatment for intrahepatic cholestasis of pregnancy (ICP). Twenty ICP patients were given UDCA at 1.5 to 2 g/d, to our knowledge the highest dosage yet reported. Effects were evaluated on conjugated bile acids (BA) in amniotic fluid (15 of 20 patients) and umbilical cord serum obtained at delivery (20 of 22 newborns), as compared with 10 untreated patients (amniotic fluid, 9 of 10 patients; cord serum, 9 of 10 newborns). Liver function tests, serum BA and UDCA were evaluated on enrollment and then weekly until 1 week after delivery. Maternal serum conjugated cholic (CCA) and chenodeoxycholic (CCDCA) acids levels fell (18.5 +/- 1.9 to 10.5 +/- 1.9 micromol/L, and 5.8 +/- 0.8 to 2.97 +/- 0.7 micromol/L, respectively [P <.01]) in treated patients, and remained unaffected (20.0 +/- 3.1 vs. 20.3 +/- 2.3, and 5.6 +/- 0.6 vs. 5.4 +/- 0.5, respectively [P = not significant]) in untreated ones. Serum conjugated UDCA levels rose to 16.5 +/- 1.8 micromol/L (P<.001). Median values of CCA and CCDCA in amniotic fluid around delivery were 4.9 +/- 12.4 and 4.8 +/- 7.7 micromol/L, respectively, in treated patients, as against 17.9 +/- 27.5 and 18.5 +/- 20.9 micromol/L in untreated ones. In treated mothers, CCA and CCDCA concentrations in cord blood were 6.0 +/- 0.9 and 5.2 +/- 0.95 micromol/L, respectively, as against 21.9 +/- 5.6 and 18.9 +/- 2.1 micromol/L in untreated ones. In treated patients, median UDCA values in amniotic fluid and cord blood were 0.8 +/- 2.4 and 0.9 +/- 0.14 micromol/L, respectively. We conclude that increasing the dose of UDCA more effectively controls ICP and improves maternal clinical outcome after delivery.

Induction of murine hepatocyte death by membrane-bound CD95 (Fas/APO-1)-ligand: characterization of an in vitro system.

Hepatocytes constitutively express CD95 (also called Fas/APO-1) and are therefore potential targets for CD95-ligand (CD95L)-mediated injury. To study this mechanism of cell death in hepatocytes we developed an in vitro model of liver cell apoptosis using membrane-bound CD95L as the inducing agent. Primary mouse hepatocytes were cocultured with NIH 3T3 fibroblasts, stably transfected with mouse CD95L (F(CD95L+)). Fibroblasts stably transfected with vector only (F(CD95L-)) served as controls. Hepatocytes from mice expressing low levels of CD95 (Fas(lpr) mice) served as controls for effects unrelated to CD95. Morphologic and biochemical studies indicate that CD95 is expressed in cultured mouse hepatocytes. Membrane-bound CD95 from transfected fibroblasts destroyed all cocultured hepatocytes within 24 hours in the absence of protein synthesis inhibitors. Characteristic features of apoptosis were observed in dying hepatocytes and occurred in the following sequence: formation of cytoplasmic blebs and nuclear condensation after 3 hours; nuclear fragmentation and DNA strand breaks after 4 hours. These changes were observed only when normal hepatocytes were cocultured with F(CD95L+) and were not observed with F(CD95L-) or in hepatocytes from Fas(lpr) mice. Anti-CD95 antibody (Jo2) evoked similar changes in hepatocytes, although to a much lesser extent. We conclude that coculture of mouse hepatocytes with F(CD95L+) is a useful in vitro model for CD95-mediated apoptosis induced by CD95L. The high incidence of apoptosis caused by membrane-bound CD95L differs from the much smaller effects induced by the Jo2 antibody. In view of the high sensitivity of hepatocytes towards CD95L we speculate that CD95L-induced liver damage in vivo may be minimized by restricting exposure of hepatocytes to CD95L.

Efficacy of ursodeoxycholic acid in association with alpha-interferon for chronic hepatitis C in alpha-interferon non-responder patients.

BACKGROUND: The aim of the present study was to evaluate the effect of combined treatment with alpha-interferon (alpha-IFN) and ursodeoxycholic acid (UDCA) on liver function tests and serum HCV-RNA in patients with chronic hepatitis C who had not responded to alpha-IFN alone. METHOD: One hundred and three patients (60 men, 43 women, mean age 49 +/- 1.3 years) who had not responded (both HCV-RNA positive and increased serum ALT levels) to 4 consecutive months of treatment with alpha-IFN (3 MU three times weekly) were randomly assigned to receive UDCA (IFN-UDCA, 53 patients, 600 mg/day) in addition to the same alpha-IFN dose, or to continue alpha-IFN alone (IFN-controls, 50 patients). After stopping alpha-IFN, patients who had received UDCA continued to receive UDCA for an additional 6-month period. The two groups were comparable for sex, basal ALT, basal yGT, genotype distribution and liver histology, while mean age was lower in controls (53 +/- 1.8 vs 46 +/- 1.8 years; P< 0.01). RESULTS: Twenty (38%) out of 53 IFN-UDCA patients had normal ALT, compared with only six (12%) out of 50 IFN-control patients (P < 0.01). HCV-RNA became undetectable in four IFN-UDCA patients. Three months after withdrawal of alpha IFN, 15 IFN-UDCA responders, but none of the IFN-controls, had normal ALT values (P< 0.01); 6 months after withdrawal, nine IFN-UDCA responders still had normal ALT (P= NS) and HCV-RNA was still undetectable in four of them. Portal and periportal inflammation showed a statistically significant improvement (Fisher's exact test P< 0.01) in IFN-UDCA patients as compared with IFN-controls, while no effect was observed on portal fibrosis. CONCLUSIONS: These data demonstrate that UDCA improves the response rate to alpha-IFN. Furthermore, in 8% of IFN-UDCA patients the response rate was sustained and associated with HCV-RNA clearance.