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The characterization (Ritter, J. K., Chen, F., Sheen, Y. Y., Tran, H. M., Kimura, S., Yeatman, M. T., and Owens, I. S. (1992) J. Biol. Chem. 267, 3257-3261) of the single-copy UGT1 gene complex encoding both bilirubin and phenol UDP-glucuronosyltransferases (transferase) has been critical to the determination of genetic defects in Crigler-Najjar Type I patients. The complex (UGT1A-UGT1G) codes for at least two bilirubin, three bilirubin-like, and two phenol transferases. Seven different exons 1, each with an upstream promoter and each encoding the amino terminus of an isoform, are arrayed in series with four common exons (encoding seven identical carboxyl termini) in the 3'-region of the locus. Predictably, a critical mutation in a common exon inactivates the entire locus. A deleterious mutation in an exon 1, as we report here for the UGT1A gene in a Crigler-Najjar Type I patient, predictably affects the amino terminus of that single isoform. The code for the predominant bilirubin isozyme, the HUG-Br1 protein, is missing the phenylalanine codon at position 170 in exon 1 of UGT1A, abolishing a conserved diphenylalanine. We demonstrate that, at the pH (7.6) routinely used for bilirubin glucuronidation studies, both the HUG-Br1 protein and human liver microsomes have approximately one-third the activity seen at the major pH optimum of 6.4 and at low ionic strength. The altered isozyme with nearly normal activity at pH 7.6 is inactive at pH 6.4, a result consistent with the definition of a pH-sensitive mutant. The Km value for bilirubin using the wild-type protein is approximately 2.5 microM at both pH 6.4 and 7.6 and that for the mutant is 5.0 microns at pH 7.6. The structure of the wild-type enzyme compared to that of the mutant indicates that hydrophobic properties at the active center are critical for metabolizing the lipophile-like substrate. The low ion/pH requirements for bilirubin glucuronidation may signal the basis for the distribution of these isozymes to an organelle (endoplasmic reticulum) that can establish compatible conditions/compartments for each catalysis.

The distribution of putative melatonin receptors in the brains of two Old World primates of the superfamily Catarrhina, Cercopithecus aethiops and Papio ursinus, was characterized using 2-[125I]iodomelatonin autoradiography. The specific binding demonstrated a discrete distribution pattern. The median eminence was intensely labelled, and examination at the light microscopic level demonstrated that the binding was confined to the small layer of cells comprising the pars tuberalis of the pituitary gland. The collar of pars distalis, present in the baboon (Papio ursinus), was diffusely labelled. No binding was detected in the pars distalis proper or the neural lobe of the pituitary gland. The binding in the suprachiasmatic nuclei was weaker, but well discernible. Diffuse faint specific binding was found in the frontal cortex and the dentate gyrus of the hippocampus. Two non-neural sites expressed strong, well-delineated binding: the walls of some brain blood vessels (the vertebral and spinal arteries, the inferior cerebellar and acoustic arteries, the basilar, pericallosal, internal carotid arteries, the arteries forming the circle of Willis) and the choroid plexuses. Binding in the arteries of the circle of Willis, the pars tuberalis and the suprachiasmatic nuclei was readily displaceable. Addition of 1 microM unlabelled 2-iodomelatonin following 45 min of preincubation with the radioactive ligand completely abrogated the binding. Co-incubation with guanosine 5'-O-(3-thiotriphosphate) led to a significant decrease in the apparent binding density in the pars tuberalis and abolished binding in the suprachiasmatic nuclei, but was without effect on the binding in the walls of the adjacent arteries, forming the circle of Willis, in the cortex and in the hippocampus. This qualitative distribution pattern demonstrates that in the two primate species studied, melatonin high-affinity, G-protein-linked binding sites are present in the pars tuberalis and the hypothalamic suprachiasmatic nuclei, and that melatonin may be acting as a synchronizer of the endogenous pacemakers' circadian activity, apart from its possible reproductive effects at the level of pars tuberalis, where the highest receptor density was observed. The strongly labelled arterial walls, and the flimsy labelled cortex and hippocampus, expressed different characteristics: though the binding was readily reversible, it was apparently not regulated by a guanine nucleotide-binding protein.

The impact of HCV infection after liver transplantation remains a topic of discussion. The aims of this study were to define the prevalence of anti-HCV antibodies in liver donors; the risk of acquired HCV infection and HCV re-infection according to the pre-transplant anti-HCV status; the prevalence of HCV infection in post-transplant chronic hepatitis. Sera from 42 recipients with follow up longer than 6 months and their donors were tested for anti-HCV. By results at pre-transplant time patients were classified as follows: donor (D) negative and recipient (R) negative (D-/R-) 31; D-/R+ 9; D+/R- 1; D+/R+ 1. Twenty-one patients with sustained hepatic dysfunction underwent liver biopsy. In group D-/R-, 5 patients showed anti-HCV positivity and 3 (9.7%) of them had acquired HCV hepatitis. In group D-/R+, 6 patients showed persistent anti-HCV positivity and 4 (44.4%) of them had recurrent HCV hepatitis; of these 2 died due to liver failure. The 2 patients of groups D+/R- and D+/R+ had normal liver function. Anti-HCV negative hepatitis was found in 2 patients. The prevalence of anti-HCV positivity in liver donors appeared low (3.2%). Acquired HCV infection rate was 9.7%. Pre-transplant HCV infection led to a high incidence of recurrence (44.4%). HCV was the major etiological agent in post-transplant chronic hepatitis (77.8%).

The prognostic value of hemostatic parameters after orthotopic liver transplantation was evaluated in 37 consecutive patients. Six simple hemostatic parameters (prothrombin time, activated partial thromboplastin time, thrombin time, thrombin coagulase time, plasma fibrinogen and platelet count) were obtained for each patient pre-transplantation and daily post-transplantation for at least 8 days. Using the results of these tests, the degree of hemostatic impairment was arbitrarily scored from 0 to 6. Starting from the first day post-transplantation, hemostatic parameters improved progressively, reaching plateau values on day 7 post-transplantation. On day 8 there were significant differences in the activated partial thromboplastin time, prothrombin time, and in the overall hemostatic scores between patients who survived at least 6 months and those who died. Comparing these hemostasis parameters with such liver function tests as AST, ALT and serum bilirubin, univariate analysis showed that activated partial thromboplastin time, coagulation score and AST were significant predictors of 6-month survival, but by multivariate analysis (Cox proportional hazard rate model) only the activated partial thromboplastin time was an independent predictor. Hence, a simple coagulation test is useful for predicting the survival of patients undergoing liver transplantation.

In Italy, many patients with B-virus (HBV) and Delta-virus (HDV) end-stage liver disease, are potential candidates to liver transplantation. Several authors have reported 70 to 100% hepatitis recurrence rates. From june 1983 to may 1989, 72 transplants on 60 patients were performed at Liver Transplantation Department of the Maggiore-Policlinico Hospital of Milan. The results in 26 HBsAg positive patients (19 with HDV infection), that underwent liver transplantation, are reported. A detailed report of 17 of these patients with a follow-up longer 7 months, and the clinical course of 4 patients with HBV reinfection, 3 of which had hepatitis recurrence, is also presented. The occurrence of HDV-RNA in the serum of 5 patients, that were negative for HBsAg and also other HBV-HDV replication markers after transplantation is discussed, together with the observation that in these patients no hepatitis recurrences or alteration of the clinical course were evident.

In order to evaluate the incidence of postoperative surgical complications requiring additional surgery, we report 73 consecutive liver orthotopic transplantations performed in 60 patients from June 1983 through June 1989. Transplantations were performed in 54 adults and 6 children for the following reasons: postnecrotic cirrhosis in 31, biliary diseases in 16, hepatobiliary malignancy in 7; Wilson's diseases in 3 and fulminant hepatitis in 3. Surgical complications requiring additional surgery occurred in 35 (58%) patients with 53 operations. Twenty-two patients (36%) had postoperative bleeding complications, 5 (8%) biliary complications, one had a late artery thrombosis and 16 (26%) had miscellaneous complications. The latter group included 6 abdominal hernias, 3 bowel perforations, 2 bowel obstructions, 2 cases of pneumothorax, 2 cases of chylous ascitis, one liver necrosis, one hepatic artery kinking, one peritonitis and one cardiac tamponade. The incidence of surgical complications was not significantly different in patients who underwent retransplantation as compared to those who had a single transplantation. We did not find a significant difference in surgical complication rate according to the preoperative liver disease. In comparison with the literature, in our series, we had a higher rate of abdominal hernia but a lower rate of biliary complications.

The influence of assay method on single dose cyclosporine (CsA) pharmacokinetics was studied in nine dogs receiving either i.v. or oral CsA. Samples were drawn from hepatic, portal, and systemic veins at various times after the dose and CsA levels were determined by radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC). Blood concentration-time data were analyzed by nonlinear least-squares regression, using two-compartment models. RIA/HPLC ratios for all samples were greater than one, and did not change significantly over time. The mean RIA/HPLC ratios for samples drawn from all three veins were higher after oral than i.v. doses of the drug (P less than 0.05). Area under the concentration-time curve (AUC) was higher and systemic clearance (CIs) lower than calculated on the basis of RIA results, regardless of the route of administration. AUC calculated for CsA metabolites (RIA-HPLC) was highest in the portal vein after an oral dose of CsA. Bioavailability was 20.4% and 27.0% when estimated using HPLC and RIA data, respectively. The mean CsA metabolite index (CMI), when calculated for hepatic, portal, or systemic vein, was greater when the drug was administered orally. The mean hepatic extraction ratio (HER) of the parent drug and for CsA metabolites was approximately 23% in i.v. and p.o. studies. These results suggest that the gastrointestinal tract may play a role in the metabolism of CsA when the drug is administered orally. In addition, if CsA metabolites not measured by HPLC have either toxic or immunosuppressive properties, the RIA assay may be more useful for monitoring patients.

A neurologically normal 3-year-old girl with Type I Crigler-Najjar syndrome was successfully treated with orthotopic liver transplantation. Preoperative serum bilirubin concentrations as high as 31 mg per dl were not diminished with phenobarbital or phototherapy. Bilirubin fractionation of duodenal bile prior to transplantation revealed 87.1% unconjugated bilirubin and 12.9% monoconjugates as determined by alkaline methanolysis-high-performance liquid chromatography. Postoperatively, the serum bilirubin concentration quickly fell to normal. Uridine diphosphate glucuronyl transferase activity in the recipient liver was not detectable. The gallbladder bile bilirubin concentration of 23.9 mg per dl was less than 15% of previously reported normal values. Since devastating kernicteric brain injury is the invariable outcome of Type I Crigler-Najjar syndrome, liver transplantation should be performed when phototherapy cannot maintain the serum bilirubin concentration at an unequivocally safe level.

Early gastric cancer was detected in 23 patients, 15 of whom had acute complications of either hemorrhage or perforation. Cases with acute complications often have a more favorable prognosis as a carcinoma may be identified while still in its pre-invasive stage. The gravity of the complication should not result in the employ of limited diagnostic procedures (e.g. endoscopy without multiple biopsies) or the use of incomplete therapeutic measures (e.g. simple suture of a perforated ulcer). The treatment of hemorrhagic gastric ulcers and perforated ulcers is discussed in greater detail. The need for a continuous follow-up of these patients with gastric ulcers is emphasized. This should include periodic endoscopy examination and multiple biopsies, even when a gastric ulcer has been apparently cured by medical treatment.

A child with the Alagille syndrome of intrahepatic bile duct paucity developed hepatocarcinoma. Disabling cirrhosis had rendered this child a suitable candidate for transplantation before the discovery of carcinoma. However, the extension of the tumor outside the liver precluded the performance of this potentially life-saving operation. Serial monitoring of the serum alpha-fetoprotein concentration may be of value in the early identification of tumors in pediatric candidates for transplantation.