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The molecular basis of capacitative (or store-operated) Ca2+ entry is still subject to debate. The transient receptor potential proteins have been hypothesized to be structural components of store-operated Ca2+ channels and recent evidence suggests that Trp3 and its closely related homolog Trp6 are gated by the N-terminal region of the inositol 1,4,5-triphosphate receptors (InsP3R). In this study, we report the existence of two isoforms of the human Trp4 protein, referred to as alpha-hTrp4 and beta-hTrp4. The shorter variant beta-hTrp4 is generated through alternative splicing and lacks the C-terminal amino acids G785-S868. Using a yeast two-hybrid assay and glutathione-S-transferase-pulldown experiments, we found that the C-terminus of alpha-hTrp4, but not of beta-hTrp4, associates in vitro with the C-terminal domain of the InsP(3) receptors type 1, 2 and 3. Thus, we describe a novel interaction between Trp proteins and InsP3R and we provide evidence suggesting that the formation of hTrp4-InsP3R complexes may be regulated by alternative splicing.

Ca(2+) signals regulate many cellular functions, including proliferation. They are governed by the inositol 1,4,5-trisphosphate receptor (IP(3)R), the only intracellular hepatic Ca(2+) channel and by the endoplasmic reticulum Ca(2+) pumps, SERCA. To characterise their role in regeneration, expression of their isoforms was studied after 2/3 hepatectomy by real-time quantitative PCR, Western blot and binding studies. We found an early increase in the expression of the IP(3)R isoform 1 which contrasted with the decrease of the expression of the IP(3)R isoforms 2 and 3 and of SERCA3. This results in a transient switch between IP(3)R isoforms 1 and 2, IP(3)R isoform 1 becoming predominant before the first round of mitosis. Binding studies detected a 30% diminution of the IP(3)R population at 24 h. In conclusion, the Ca(2+) signalling machinery is regulated, after hepatectomy, by changes in expression of the IP(3)R and SERCA isoforms to adapt Ca(2+) signals to the regenerative state., (Copyright 2000 Academic Press.)

Ca(2+) signals mediate the hepatic effects of numerous hormones and growth factors. Hepatic Ca(2+) signals are elicited by the inositol trisphosphate receptor, an intracellular Ca(2+) channel. Three isoforms of this receptor have been identified; they are expressed and regulated differently. We investigated the effect of liver fibrosis and cirrhosis on the hepatic expression of the inositol trisphosphate receptor isoforms. Two different rat models were used: bile duct ligation (fibrosis) and chronic exposure to CCl(4)/phenobarbital (cirrhosis). Messenger RNA levels were determined by ribonuclease protection assay (RPA), competitive polymerase chain reaction (PCR) followed by Southern blotting, and real-time quantitative PCR. Protein expression was assessed by Western blotting; tissue distribution was assessed by immunohistology. In control animals, isoform 2 was the predominant isoform, isoform 1 represented less than one third, and isoform 3 less than 1%. After bile duct ligation, expression of types 1 and 3 increased 1.9- and 5.7-fold, and expression of type 2 decreased 2. 5-fold at the protein level. After exposure to CCl(4)/phenobarbital, expression of types 1, 2, and 3 were 2.4-, 0.9-, and 4.2-fold their expression in control animals. Type 2 was localized to the apical domain of hepatocytes, consistent with a role for Ca(2+) signals in canalicular function. Type 3 was detectable in intrahepatic bile duct epithelial cells and not in hepatocytes, suggesting that Ca(2+) signals may be regulated differently in these cells. Signaling through inositol trisphosphate receptor participates in the pathogenesis of cirrhosis, because this process affects the expression of its isoforms.

Background: Graft rejection and infection remain major problems following liver transplantation; both are heavily influenced by the immunosuppressive regimen. Despite the disparity in the primary disease leading to transplantation, all patients receive the same posttransplant immunosuppressive treatment in a given center. The aim of this study is to detect a possible effect of the underlying disease on the incidence of early acute rejection episodes after orthotopic liver transplantation (OLT)., Patients and Methods: Retrospective analysis on all 101 consecutive liver transplants performed in 95 patients between 1983 and March 1998; five of these patients, surviving less than 30 days, were not included. The immunosuppressive regimen was based on conventional triple therapy during the whole study period. The diagnosis and treatment of acute rejection within the first 30 days post-OLT was uniform throughout the whole study period., Results: Though there were no differences with respect to patients' characteristics [age, child classification, number of HLA-mismatches or cytomegalovirus (CMV)-serocompatibility], patients with primary biliary cirrhosis (PBC) showed a significant increase of acute rejection after OLT compared with the other patients transplanted for other liver diseases (P = 0.024). The incidence of infection was not elevated in patients transplanted for PBC when compared with other diagnoses., Conclusion: Our results indicate that primary liver disease may be a determinant for acute graft rejection in PBC. Furthermore, these results suggest that immunosuppressive regimens based on the underlying disease should be considered.

Cirrhosis occurs in 20-50% of patients with hepatitis C and is thought to be irreversible. We describe two patients with cirrhosis secondary to hepatitis C in whom the extensive fibrosis and cirrhosis appeared to regress in response to treatment with interferon-alpha (IFN-alpha). Both patients were in the early stages of cirrhosis, class A in the Child-Pugh classification, total score 5 for each patient. Both responded fully to IFN-alpha and had normalization of all liver function tests and disappearance of hepatitis C viral RNA. Liver biopsies, performed before and after treatment, were coded unpaired by patient, combined with 21 liver biopsies from eight other patients with chronic hepatitis, and read independently by two pathologists using the Knodell scoring system. Knodell scores decreased from 14 to 3.5 and from 13.5 to 4 in these two patients. Cirrhosis and extensive fibrosis, present at baseline, were not present on follow-up liver biopsies, which were of sufficient size to reduce the likelihood of sampling error. We conclude that hepatic fibrosis and clinically early cirrhosis may be reversible in some patients with hepatitis C who respond to treatment with IFN-alpha.

Background & Aims: Guanosine 3',5'-cyclic monophosphate (cGMP), whose production is stimulated by the interaction of nitric oxide, natriuretic peptides, and guanylin with their respective guanylate cyclases, activates secretion through ion channels in several epithelia. Cl- channels have been identified in the apical membrane of biliary epithelial cells. The aim of this study was to investigate the production of cGMP and its effects on Cl- permeability in biliary epithelial cells., Methods: Halide efflux measurement, whole-cell patch clamp recording, radioimmunoassay, and reverse-transcription polymerase chain reaction using two human biliary cell lines (H69 and Mz-ChA-1) were performed., Results: In cells equilibrated with 125I, bromo-cGMP stimulated halide efflux by 22%. In whole-cell patch clamp recordings, the addition of cGMP intracellularly, or of atrial natriuretic peptide extracellularly, stimulated inward currents at negative membrane potentials, consistent with Cl- efflux through open channels. In H69 cells, atrial and C-type natriuretic peptides stimulated production of cGMP. Mz-ChA-1 responded only to atrial natriuretic peptide. Both cell lines expressed messenger RNA for the guanylate cyclase type A receptor and the guanylate cyclase free-clearance receptor., Conclusions: These data suggest that natriuretic peptide stimulates cGMP production in human biliary epithelial cells, which in turn may regulate ductular bile formation through the opening of Cl- channels.

Background: Hepatic fibrosis and cirrhosis occur in many types of chronic liver injury and generally seem to be irreversible., Objective: To determine whether cirrhosis caused by autoimmune hepatitis can be reversible., Design: Retrospective study., Patients: Eight patients with autoimmune hepatitis and cirrhosis who responded to medical therapy and had follow-up liver biopsy while in clinical and biochemical remission., Measurements: Biopsy specimens were randomly coded in an unpaired manner according to patient and were read independently by two pathologists using the Knodell scoring system., Results: The median alanine aminotransferase level decreased from 10.30 mukat/L to 0.37 mukat/L, the median serum bilirubin level decreased from 70 mumol/L to 10 mumol/L, and the median serum albumin level increased from 34 g/L to 43 g/L. Cirrhosis, extensive fibrosis, or both were present in all patients at diagnosis but were not present on follow-up liver biopsy. The median Knodell score decreased from 14.0 to 1.3, and the median fibrosis score decreased from 3.3 to 0.8., Conclusion: Hepatic fibrosis and cirrhosis may be reversible in some patients in whom autoimmune hepatitis responds to treatment.

The bile canaliculi of hepatocytes contract spontaneously, and it is hypothesized that this canalicular motility provides a propulsive force for normal intrahepatic bile flow. Cytochalasin disrupts actin polymerization, inhibits contraction, and decreases bile flow. We investigated whether this cholestasis was associated with impaired canalicular secretion. Isolated rat hepatocyte doublets, with and without incubation with 2 mumol/L cytochalasin D (cytD), were superfused, under first-order conditions, to steady state with fluorescein isothiocyanate-labeled glycocholic acid (FITC-GC) and carboxy-4',5'-dimethylfluorescein diacetate (CMFD), which are fluorescent substrates for the bile acid and the nonbile acid organic anion transport pathways, respectively. Fluorescent microscopic images were quantified and the data analyzed by noncompartmental and compartmental kinetic methods. cytD dilated the canalicular spaces fivefold but did not change the proportion of doublets that secreted either probe. Cytochalasin did not affect the mean cellular transit times of FITC-GC (2.8 and 2.5 minutes for control and cytochalasin-treated groups, respectively) and of carboxy-4',5'-dimethylfluorescein (3.8 and 3.7 minutes, respectively). Analysis with a three-compartment model gave estimates of the rate constants for canalicular secretion: 0.21 +/- 0.04 and 0.22 +/- 0.03 min-1 in control and treated cells, respectively, for FITC-GC, and 0.14 +/- 0.01 and 0.16 +/- 0.02 min-1, respectively, for carboxy-dimethylfluorescein. When kinetics are first-order, the canalicular secretion of organic anions is not altered by actin disruptive agents, suggesting that actin filaments do not modulate the function or distribution of these transporters. This suggests that impaired contractility rather than impaired canalicular secretion is the mechanism of cytD-induced cholestasis.

The regulation of the inositol 1,4,5-trisphosphate (IP3) receptor in liver was analyzed using a novel superfusion method. Hepatic microsomes were loaded with 45Ca2+, and superfused at high flow rates to provide precise control over IP3 and Ca2+ concentrations ([Ca2+]) and to isolate 45Ca2+ release from reuptake. 45Ca2+ release was dependent on both [Ca2+] and IP3. The initial rate of 45Ca2+ release was a biphasic function of [Ca2+], increasing as [Ca2+] approached 3 microM but decreasing at higher concentrations, suggesting that the hepatic IP3 receptor is regulated by [Ca2+] at two sites, a high affinity potentiation site and a low affinity inhibitory site. The relationship between initial rates and IP3 concentration was steep (Hill coefficient of 3.4), suggesting that activation of the calcium channel requires binding of at least 3 IP3 molecules. IP3 concentrations above 10 microM produced rapid decay of release rates, suggesting receptor inactivation. Superfusion with 10 microM IP3 under conditions that minimize calcium release ([Ca2+] < 1 nM) inhibited 45Ca2+ release in response to subsequent stimulation (400 nM Ca2+). These data suggest sequential positive and negative regulation of the hepatic IP3 receptor by cytosolic calcium and by IP3, which may underlie hepatocellular propagation of regenerative, oscillatory calcium signals.

Between 1977 and 1994, our center administered successively 4 different chemotherapy regimens to 242 evaluable patients with locally advanced breast cancer. Patients with inflammatory signs were excluded. Sixty-eight patients were treated by AVCF (A (adriamycine) + V (vincristine) + C (cytoxan) + F (5FU)), 47 by AECF (A + E (vindesine) + C + F), 81 by CAFP (C + A + F + P (prednisone)) and 46 by AN (A + N (vinorelbine)). The mean number of cycle was 3. One hundred and twenty-five patients (52.5%) responded to chemotherapy and we recorded 35 complete response (14.7%). The response rates at the different combinations were respectively: AVCF: 29.4%, AECF: 53.2%, CAFP: 64.9%, AN: 65.2%, and were independent of tumor size, grade and receptor status. The response rate at the AVCF regimen was significantly worse than the others (p = 0.0005). Breast conserving surgery was performed in 31 patients (14%) and 17 patients (8%) had a complete response. Among the 35 patients with complete response, 21 were treated by radiotherapy alone. Local recurrence occurred in 19 patients (7.9%) and 96 (40%) had advanced disease. The mean follow-up of AVCF regimen was 150 months, 115 months for AECF, 111 for CAFP and 42 months for AN. The disease-free survival and the overall survival were significantly better with AECF, CAFP and AN regimens (DFS p < 0.04, OS p < 0.02). Survival was better in those patients with an objective response (p = 0.002) or with non-affected axillary node at the time of surgery. Our study showed already that AVCF combination was significantly lower than AECF, CAFP, AN in terms of response rate, disease-free survival and overall survival. Waiting the results of randomized studies about the impact of neoadjuvant chemotherapy on survival, we look for chemotherapy regimen improving the rate of conservative surgery.

The second messenger, cyclic guanosine monophosphate (cGMP), mediates the actions of nitric oxide, natriuretic peptides, and microbial toxins on cellular contractility and electrolyte movement. Because both hepatocellular contractility and electrolyte secretion participate in bile formation, we investigated the actions of cGMP on this process in intact liver. In rat liver perfused with 8-bromo-cyclic GMP (bcGMP) at 0.5 and 3 micromol/min, bile flow increased by 5% and 31%, respectively. The biliary excretion of the bile acid, taurocholate ([3H]-labeled; 1 micromol/min) and of the organic anion, bromosulfophthalein ([35S]-labeled; tracer dose), was unchanged. The paracellular and transcytotic pathways of biliary excretion, assessed by horseradish peroxidase (HRP), were unaffected. BcGMP was concentratively secreted into bile and the accompanying 30% increase in the biliary clearance of erythritol suggested that the choleresis was primarily osmotic in nature. Unlike cyclic adenosine monophosphate (cAMP), which stimulates bile acid dependent bile flow and transcytosis, bcGMP increased bile acid independent bile flow mainly as a result of its concentrative biliary secretion.

Background/aims: The biochemical mechanism of bile canalicular contraction is similar to that of smooth muscle contraction. Contraction follows inositol-1,4,5-trisphosphate (InsP3)-dependent Ca2+ release, which activates actin-myosin interactions. Nitric oxide is a myorelaxant through the actions of 5'-cyclic guanosine monophosphate (cGMP) and is produced in hepatocytes exposed to endotoxin and cytokines. The aim of this study was to investigate the effect of nitric oxide on canalicular contraction and to determine the mechanism by which cGMP interferes with the contractile signal., Methods: The canalicular motility in rat hepatocyte doublets was measured by microscopic image analysis, and intracellular Ca2+ was measured by fluorescence microscopy. cGMP and InsP3 were determined by radio-immunoassay and high-pressure liquid chromatography. Ca2+ release from liver homogenate was measured by filtration and superfusion assays., Results: Compounds that release nitric oxide stimulated hepatocellular production of cGMP and prevented agonist-induced contraction by inhibiting the increase in intracellular Ca2+. The cGMP analogue bromo-cGMP prevented contraction and the increase in Ca2+. Bromo-cGMP marginally decreased InsP3 production. cGMP blocked InsP3-dependent Ca2+ release from internal stores., Conclusions: These findings suggest that nitric oxide interferes with Ca2+ signals by cGMP-mediated inhibition of the InsP3 receptor/Ca2+ channel and that hepatocellular production of nitric oxide may be cholestatic by impairing canalicular motility.

To investigate the potential role of lysosomes in cirrhosis, the activity of lysosomal enzymes was analyzed in rats with cirrhosis induced by bile-duct ligation. Twenty-eight days after surgery, the activity of lysosomal enzymes was markedly increased in the homogenate of cirrhotic livers (e.g. arylsulfatase 7 +/- SD 1 vs 17 +/- 3 nmol.min-1.mg-1 in controls and cirrhotics, respectively; p < 0.001). The corresponding plasma levels were also increased (arylsulfatase: 10 +/- 1 vs 25 +/- 9 pmol.min-1.mg-1; p < 0.01). In contrast, the activities of these enzymes in lysosomal fractions did not differ, suggesting an increase in number of lysosomes. The increased lysosomal activity correlated with severity of cirrhosis as assessed by the aminopyrine breath test and with cholestatic parameters but less with transaminases. Since macrophages, cells which are rich in lysosomes, could contribute to the increase in lysosomal enzyme content, these cells were estimated stereologically after being marked immunohistochemically with a monoclonal antibody against the rat macrophage membrane antigen ED2. ED2 positive cells were increased 2.7-fold in cirrhotic livers. This increase cannot account for the observed increase in hepatic lysosomal enzyme content. Furthermore, 1 week after bile-duct ligation, when there was cholestasis but not yet cirrhosis, lysosomal enzyme activities were already increased. These data support the idea that the increased hepatic lysosomal activity in biliary cirrhosis is of hepatocyte rather than of macrophage origin, and is presumably related to cholestasis.

On routine blood screens, persistent conjugated hyperbilirubinemia was discovered in two groups of closely related adult female golden lion tamarins (Leontopithecus rosalia rosalia, n = 8). Bromosulfophthalein (BSP) retention tests were performed on four hyperbilirubinemic and three control tamarins. BSP excretion was delayed in hyperbilirubinemic tamarins as compared with controls. Grossly, liver of affected tamarins was dark brown to black, with a prominent reticulated pattern. Histologic examination revealed abundant intrahepatic pigment, primarily in a centrilobular and midzonal distribution. Most of the pigment did not react with Perls' Prussian blue method for iron, Hall's method for bilirubin, or the Armed Forces Institute of Pathology acid-fast method for lipofuscin but was positive with Fontana and lipofuscin-ferric ferricyanide reduction techniques. Liver from control golden lion tamarins had intrahepatocellular Perls' iron-positive pigment diffusely throughout the lobule with a small amount of Fontana method-positive pigment. Ultrastructurally, hepatocytes from a hyperbilirubinemic tamarin contained pleomorphic electron-dense structures within lysosomes. Transport studies demonstrated secretion of fluorescein isothiocyanate-labeled glycocholic acid, a fluorescent bile acid analog, into bile canaliculi and no secretion of carboxydichlorofluorescein diacetate, a non-bile acid organic anion, by liver from a hyperbilirubinemic tamarin. In contrast, control liver secreted carboxydichlorofluorescein diacetate readily into bile canaliculi. The clinicopathologic presentation of this syndrome in golden lion tamarins is similar to that described for Dubin-Johnson syndrome of human beings.

To investigate the potential role of lysosomes in cirrhosis, we analyzed the activity of lysosomal enzymes in rats exposed long-term to phenobarbital and carbon tetrachloride. The activity of lysosomal enzymes was markedly increased in the homogenate of cirrhotic livers (e.g., arylsulfatase 9 +/- S.D.2 vs. 16 +/- 6 nmoles.min-1.mg-1 in control rats and cirrhotic rats, respectively; p less than 0.001). The corresponding plasma levels were also increased (7 +/- 1 vs. 12 +/- 3 nmoles.min-1.mg-1; p less than 0.01), whereas biliary excretion was diminished (16 +/- 7 vs. 7 +/- 2 pmol.min-1.gm liver-1; p less than 0.05) in cirrhotic rats. Stereological quantification of lysosomes visualized cytochemically revealed an increase of pericanalicular lysosomes averaging 1.5 +/- 0.4 around a canaliculus in controls and 3.7 +/- 1.0 in cirrhotic rats (p less than 0.01). Because this suggested a defect in the transcellular vesicular pathway, we investigated the biliary excretion of horseradish peroxidase and epidermal growth factor in perfused livers. Bile flow and total horseradish peroxidase excretion were similar in control rats and cirrhotic rats. However, the early peak of biliary horseradish peroxidase excretion--usually taken as evidence of paracellular transport--was increased in cirrhotic rats (13 +/- 7 vs. 57 +/- 22%; p less than 0.01), whereas the second peak--reflecting the transcellular vesicular pathway(s)--was markedly reduced (87 +/- 7 vs. 43 +/- 22%; p less than 0.001). A similar reduction in the biliary excretion of intact epidermal growth factor and of its degradation products was found. These results demonstrate an increased number of lysosomes in hepatocytes of cirrhotic livers; this appears to be the result of accumulation rather than proliferation, in view of the reduced transcellular vesicular movement of different markers into bile.

Quantitative liver function tests are based on the clearance concept and measure the plasma disappearance of a test compound such as galactose. Metabolism is inferred to be predominantly hepatic, and usually no knowledge is obtained of the true time course of metabolite formation. Dynamic 31phosphorus magnetic resonance spectroscopy after intravenous administration of fructose directly measures hepatic sugar metabolism. To determine the feasibility and the utility of 31P magnetic resonance spectroscopy, we studied the responses of six healthy subjects and nine patients with nonalcoholic cirrhosis to a fructose load. Results were related to the impairment of hepatic function assessed by the galactose-elimination capacity test. Liver spectra were acquired in a 1.5 T whole-body nuclear magnetic resonance unit with a surface coil (9-cm diameter) placed ventrally on the liver; the one-dimensional chemical-shift imaging technique was used to obtain spectra from tissue slices parallel to the surface coil. After a basal spectrum had been obtained, fructose (250 mg/kg) was injected intravenously, and further spectra were collected sequentially every 6 min for 1 hr. Formation of monophosphate esters (9% +/- 5% vs. 20% +/- 8% of total area; p less than 0.01) and utilization of inorganic phosphate (5% +/- 4% vs. 11% +/- 3% of total area; p less than 0.005) were markedly decreased in cirrhotic patients. These measures correlated with the severity of the impairment of liver function measured by the galactose-elimination capacity (r = 0.53 to 0.69; p less than 0.05). We conclude that dynamic 31P magnetic resonance spectroscopy is a safe, clinically feasible test that allows detailed insights into biochemical events in liver disease.

We review the 257 patients hospitalized for meningitis in the Cantonal University Hospital, Geneva between 1st January 1980 and 31st December 1986. 104 patients had acute bacterial meningitis (32 Str. pneumoniae, 21 N. meningitidis, 10 Listeria monocytogenes, 8 streptococci, 5 H. influenzae, 5 staphylococci, 4 gram negative bacilli and 19 without identified bacteria), 124 patients had viral meningitis and 29 meningitis of other etiologies (6 tuberculous meningitis, 2 fungal meningitis, 1 leptospiral meningitis, 5 neoplastic meningitis--one already counted because of a meningitis due to Staph. epidermidis--2 meningitis consecutive to a meningeal irritation, 4 already treated meningitis of undetermined etiology, 2 chronic meningitis and 8 meningoencephalitis). The total mortality was 14.4%. It was zero in viral meningitis and 28% in bacterial meningitis (47% in cases of Str. pneumoniae, 5% in cases of N. meningitidis, 20% in cases of Listeria monocytogenes, 38% in cases of streptococci, 0% in cases of H. influenzae, 60% in cases of staphylococci, 50% in cases of gram negative bacilli, 16% in cases of unidentified bacteria). The striking difference in mortality emphasizes the importance of recognizing a bacterial etiology in order to institute antibiotic therapy as soon as possible. The delay between admission and lumbar puncture averaged 15 hours (range 0.25-96 h) in patients with acute bacterial meningitis and 6.3 hours (0.5-80 h) in patients with viral meningitis. The delay between admission and institution of the antibiotics averaged 5.3 hours (1-48 h) in cases of acute bacterial meningitis and 4.8 hours (0.5-48 h) in cases of viral meningitis. A better clinical workup may provide a reliable diagnosis sooner. In the collective with bacterial and viral meningitis headaches, fever or nuchal rigidity were present in over 80% of the cases. The following features were significantly associated with a bacterial etiology: age over 30 years, alcoholism, concomitant neoplasm, cough, coma, pulmonary rales, new neurological signs or petechia. At least one of these 4 latter signs was present in more than 70% of the cases with acute bacterial meningitis compared to 6% in cases of viral meningitis. Thus the clinical presentation alone serves to recognize the meningitis and to differentiate between a bacterial or viral etiology, thus permitting an immediate therapeutic decision without waiting for complementary investigations. The 104 patients with acute bacterial meningitis were treated with antibiotics: 60 with penicillin, 17 with ampicillin and 26 with other antibiotics; one case did not receive antibiotics. More than the half of the cases with viral meningitis have got antibiotics (52%).