Gene replacement therapy for genetic hepatocellular jaundice.

Jaundice results from the systemic accumulation of bilirubin, the final product of the catabolism of haem. Inherited liver disorders of bilirubin metabolism and transport can result in reduced hepatic uptake, conjugation or biliary secretion of bilirubin. In patients with Rotor syndrome, bilirubin (re)uptake is impaired due to the deficiency of two basolateral/sinusoidal hepatocellular membrane proteins, organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3. Dubin-Johnson syndrome is caused by a defect in the ATP-dependent canalicular transporter, multidrug resistance-associated protein 2 (MRP2), which mediates the export of conjugated bilirubin into bile. Both disorders are benign and not progressive and are characterised by elevated serum levels of mainly conjugated bilirubin. Uridine diphospho-glucuronosyl transferase 1A1 (UGT1A1) is responsible for the glucuronidation of bilirubin; deficiency of this enzyme results in unconjugated hyperbilirubinaemia. Gilbert syndrome is the mild and benign form of inherited unconjugated hyperbilirubinaemia and is mostly caused by reduced promoter activity of the UGT1A1 gene. Crigler-Najjar syndrome is the severe inherited form of unconjugated hyperbilirubinaemia due to mutations in the UGT1A1 gene, which can cause kernicterus early in life and can be even lethal when left untreated. Due to major disadvantages of the current standard treatments for Crigler-Najjar syndrome, phototherapy and liver transplantation, new effective therapeutic strategies are under development. Here, we review the clinical features, pathophysiology and genetic background of these inherited disorders of bilirubin metabolism and transport. We also discuss the upcoming treatment option of viral gene therapy for genetic disorders such as Crigler-Najjar syndrome and the possible immunological consequences of this therapy.

Hereditary hyperbilirubinemias.

Inherited disorders of bilirubin metabolism involve four autosomal recessive syndromes: Gilbert, CriglerNajjar, Dubin-Johnson and Rotor, among which the first two are characterized by unconjugated and the second two by conjugated hyperbilirubinemia. Gilbert syndrome occurs in 2%-10% of general population, while others are rare. Except for Crigler-Najjar syndrome, hereditary hyperbilirubinemias belong to benign disorders and thus no treatment is required.

[Hereditary pigmentary hepatoses (functional hyperbilirubinemias)].

Reviewed in this paper are bilirubin metabolism, definition and classification of hereditary pigmentary hepatoses, their pathogenesis and forms of inheritance, clinical picture, laboratory, instrumental and differential diagnosis, morphological changes in the hepatic tissue, and therapeutic strategies.

The patient presenting with isolated hyperbilirubinemia.

Hyperbilirubinemia is a common laboratory finding in clinical practice, being found in several haematological and liver diseases as well as in familial conditions (5-10% in Western countries). Although most of the familial forms of hyperbilirubinemia are classically viewed as benign conditions, they have gained an increased interest in the last few years since recent data have indicated that subjects with an impaired bilirubin metabolism may have an increased susceptibility to drug toxicity. The authors briefly review the main steps of bilirubin metabolism, with a special emphasis on the emerging concepts on the molecular mechanisms of regulation by nuclear receptors (NRs) and genetic factors. Then the different forms of isolated hyperbilirubinemia occurring in both adults and paediatrics are systematically analysed, and a new categorisation is also proposed in light of the recent advances in bilirubin research. Finally, a diagnostic algorithm is discussed, along with a correct approach to its management, in order to avoid unnecessary medical investigations.

Liver repopulation with bone marrow derived cells improves the metabolic disorder in the Gunn rat.

BACKGROUND: Reversible ischaemia/reperfusion (I/R) liver injury has been used to induce engraftment and hepatic parenchymal differentiation of exogenous beta2-microglubulin(-)/Thy1(+) bone marrow derived cells. AIM: To test the ability of this method of hepatic parenchymal repopulation, theoretically applicable to clinical practice, to correct the metabolic disorder in a rat model of congenital hyperbilirubinaemia. METHODS AND RESULTS: Analysis by confocal laser microscopy of fluorescence labelled cells and by immunohistochemistry for beta2-microglubulin, 72 hours after intraportal delivery, showed engraftment of infused cells in liver parenchyma of rats with I/R, but not in control animals with non-injured liver. Transplantation of bone marrow derived cells obtained from GFP-transgenic rats into Lewis rats resulted in the presence of up to 20% of GFP positive hepatocytes in I/R liver lobes after one month. The repopulation rate was proportional to the number of transplanted cells. Infusion of GFP negative bone marrow derived cells into GFP positive transgenic rats resulted in the appearance of GFP negative hepatocytes, suggesting that the main mechanism underlying parenchymal repopulation was differentiation rather than cell fusion. Transplantation of wild type bone marrow derived cells into hyperbilirubinaemic Gunn rats with deficient bilirubin conjugation after I/R damage resulted in 30% decrease in serum bilirubin, the appearance of bilirubin conjugates in bile, and the expression of normal UDP-glucuronyltransferase enzyme evaluated by polymerase chain reaction. CONCLUSIONS: I/R injury induced hepatic parenchymal engraftment and differentiation into hepatocyte-like cells of bone marrow derived cells. Transplantation of bone marrow derived cells from non-affected animals resulted in the partial correction of hyperbilirubinaemia in the Gunn rat.

New concepts in bilirubin and jaundice: report of the Third International Bilirubin Workshop, April 6-8, 1995, Trieste, Italy.

The workshop covered three major areas: Unconjugated bilirubin (UCB) chemistry and physical chemistry; UCB transport and intracellular trafficking; and evaluation and therapy of neonatal and congenital hyperbilirubinemias. Findings of studies in the chemistry and physical chemistry area were as follows. (1) Nuclear magnetic resonance (NMR) studies of highly enriched 13COOH mesobilirubin in water-dimethyl sulfoxide systems indicated that the pKa values of the carboxyl groups are 4.2 and 4.9, respectively. This finding differs from some reports that suggest that the two pKa values in aqueous systems are near or above pH 7.0. (2) Contrasting views of the hydrophobic interactions of UCB with bile salts were presented: one suggested that multiple bile salt monomers bind to one UCB molecule; the other suggested that UCB binds to the nonpolar surface of helical bile salt micelles. (3) Structures were proposed for the varied calcium and copper bilirubinate salts formed at various pH values and cation/UCB ratios. (4) Studies of binding of UCB to human serum albumin (HSA) showed marked diminution of UCB-binding affinity as albumin and chloride concentrations increased. (5) A unique UCB derivative, bilirubin-C10-sulfonic acid, was identified as the major bile pigment in bullfrog bile. (6) New methods were presented for removal of impurities from preparations of bile salts and UCB. Findings of studies in the transport area were as follows. (1) Four putative basolateral and two putative canalicular hepatocytic transporters of UCB and related organic anions were described. Special emphasis was given to the adenosine triphosphate (ATP)-dependent canalicular multi-specific organic anion transporter that is defective in three strains of mutant rats with congenital conjugated hyperbilirubinemia. (2) The roles of the classical and newer molecular biological approaches to identification of these transporters were contrasted, and their limitations were discussed. (3) The relative roles of the multiple carriers in UCB transport under different conditions and substrate concentrations were discussed. (4) Cytosolic UCB-binding proteins (e.g., ligandin) were shown to promote transcellular movement of UCB by solubilizing and transporting the pigment in the aqueous phase while limiting binding of UCB to the relatively immobile membranes of cell organelles. (5) Mechanisms were presented for translocation of UDP-glucuronic acid (UDPGA) into the lumenal location of UDPGA transferase in the endoplasmic reticulum, as well as the enhancement of this process by N-acetyl-glucosamine. Studies in the neonatal and congenital jaundice area were as follows. (1) Criteria were reviewed for initiating treatment of neonatal jaundice, emphasizing the primacy of serum bilirubin levels, gestational age, and hemolysis as risk factors for kernicterus. (2) New methods were presented for frequent, automated monitoring of serum bilirubin levels and breath CO levels as an index of rates of formation of UCB from heme. (3) The current status and limitations of new approaches to treatment of severe unconjugated hyperbilirubinemia were discussed: hepatocyte transplantation and gene therapy, still in the stage of development in animal models, have provided only partial and temporary relief of hyperbilirubinemia; extracorporeal liver assist devices have had some success in initial human studies; and inhibition of heme oxygenase (HO) with metalloporphyrins, especially tin mesoporphyrin, which markedly decreases bilirubin production for prolonged periods, is a new alternative to phototherapy. (4) The ontogeny of the two HO isozymes was contrasted in the liver, spleen, kidney, and lung.

Impact of hyperbilirubinaemia and transient mother-child separation in the neonatal period on mother-child attachment in the 1st year of life.

Hyperbilirubinaemia (HyB) is the most common health disturbance in the neonatal period. The aim of this prospective study is to determine whether HyB and/or phototherapy (PhT) together with transient separation during the neonatal period are associated with impaired mother-child attachment after the 1st year of life. We divided 107 healthy term infants into three groups: 29 markedly icteric infants who underwent PhT (mother-child separation), 40 mildly icteric infants without PhT and a control group of 38 nonicteric infants. At the age of 1 year a paediatric examination and a Denver test were performed, and the mother-infants pairs were observed in Ainsworth's strange situation. The results show a similar distribution of the attachment patterns in the three different groups of infants. HyB and PhT do not negatively seem to affect the quality of attachment. Analysis of additional aspects showed that maternal coping and her perception of the child appear to be more important antecedents of the quality of attachment after the 1st year of life.

Hepatocytes immobilised by microencapsulation in artificial cells: effects on hyperbilirubinemia in Gunn rats.

The possibility of using hepatocytes encapsulated in a calcium-alginate-polylysine matrix to lower bilirubin levels in hyperbilirubinemia was investigated. The animal model was the Gunn rat. The microencapsulated hepatocytes were injected intraperitoneally. 15 X 10(6) microencapsulated hepatocytes from Wistar rats, lowered the bilirubin from 14 mg/100 ml to 6 mg/100 ml after 20 days. The bilirubin is still depressed after 90 days. After encapsulation, Sprague-Dawley hepatocytes were as effective as free hepatocytes in lowering bilirubin levels in Gunn rats. After 68 days, the free Sprague-Dawley hepatocytes were not rejected.

Multiple plasma exchanges successfully maintain a young adult patient with Crigler-Najjar syndrome type I.

Plasmapheresis has been shown to reduce total and free bilirubin levels in acute exacerbations of Crigler-Najjar syndrome, type I (CNS-TI), but its effectiveness in long-term management has not been reported. An 18-year-old (yo) male with CNS-TI, who required prolonged daily high-intensity phototherapy to prevent cerebral nervous system symptoms, developed increasingly frequent bouts of confusion, nausea, and vomiting associated with free bilirubin concentrations (fbcs) greater than 10-15 nmol/L. Pending consideration of orthotopic liver transplantation, plasma exchange (approximately 3 liters per procedure) was begun in 12/84 using the IBM/COBE 2997 with 5% albumin as replacement fluid. Frequency of treatments was guided by twice weekly fbcs, with plasma exchange for fbc greater than 10-15 nmol/L. Pre-exchange and postexchange fbcs ranged from 27.5 to 11 nmol/L and 9.2 to 2 nmol/L, respectively. Seventy-two exchanges were performed over a 28 month period. Irreversible CNS damage did not occur, and the patient underwent successful liver transplantation in April of 1987, with complete correction of his metabolic disorder. He remains well 18 months following transplantation.

Crigler-Najjar syndrome: treatment at home with phototherapy.

An infant became jaundiced in the neonatal period. The serum bilirubin failed to fall with phototherapy. A diagnosis of Crigler Najjar type 1 syndrome was made by exclusion and confirmed by liver biopsy. The infant has been successfully treated at home with phototherapy. Liver transplantation remains a therapeutic option.

Transplantation of isolated hepatocytes into the pancreas.

In previous research into hepatocyte transplantation (HTX) the spleen was the preferred acceptor organ for isolated donor hepatocytes. In this study the pancreas was tested as an acceptor organ for HTX. HTX into the pancreas or spleen was performed by injection of 10(7) isolated hepatocytes into the parenchyma of these organs. Intrapancreatic hepatocytes showed good viability 3 months after syngenic HTX as assessed by histological and immunocytochemical parameters. Definite proof of sustained metabolic activity of normal hepatocytes, 3 months after transplantation into the pancreas of congenitally jaundiced rats, was obtained by demonstration of bilirubin conjugates in bile of the recipients: 4.0% of total biliary bilirubin was conjugated. Intrasplenic HTX, however, was more effective and resulted in a conjugated fraction of 17.7% of total biliary bilirubin (p less than 0.001). Reduction of total plasma bilirubin was significant with both methods, but more pronounced in intra-splenic HTX. Bile drainage from the hepatocellular transplant via the pancreatic excretory system into the gut was not observed: conjugated bilirubins were not found in pancreatic juice of HTX-treated jaundiced rats. Intrapancreatic HTX did not adversely affect the host rat; evidence of pancreatitis or diabetes was not found. It is concluded that the pancreas is a suitable acceptor organ for HTX. However, intrapancreatic HTX appears to be less effective than intrasplenic HTX in the treatment of enzyme deficiency disease.

Hepatobiliary excretion of organic anions in double-mutant rats with a combination of defective canalicular transport and uridine 5'-diphosphate-glucuronyltransferase deficiency.

Mutant rats with a selective defect for the hepatobiliary excretion of organic anions (GT+TR- rats) are valuable models to study hepatic transport processes. However, retained conjugates in the livers of these rats may secondarily affect hepatic uptake, metabolism, and excretion of other compounds and this may confound the interpretation of test results. We have developed double mutants (GT-TR-) rats with both a conjugation and an excretion defect by cross-breeding uridine 5'-diphosphate-glucuronyl-transferase-deficient GT-TR+ Gunn rats with transport-deficient GT+TR- rats. Phenotypically, GT-TR- rats and Gunn rats are alike in that both have unconjugated hyperbilirubinemia. Intravenous administration of tetrabromosulphthalein, bilirubin diglucuronide, and bilirubin monoglucuronide revealed a significant difference in that the clearance of these compounds was reduced to 10%, 10%, and 20%, respectively, in GT-TR- rats when compared with Gunn rats. The hepatic elimination of tetrabromosulphthalein in GT-TR- rats and in GT+TR- rats is impaired to the same extent. Thus, both have a similar hepatic excretion defect. However, bile flow and bile acid excretion in GT+TR- rats are more depressed than in GT-TR- rats: bile flow, 88 +/- 3 vs. 36 +/- 1 microliters/min.kg and bile acid excretion, 3.4 +/- 0.2 vs. 1.5 +/- 0.1 mumol/min.kg in GT-TR- and GT+TR- rats, respectively. This suggests that accumulated glucuronides in the liver inhibit bile flow and bile acid excretion. To test whether conjugated bilirubin and the photoisomers of unconjugated bilirubin are excreted via the same transport pathways, the effect of phototherapy was studied in GT-TR- rats and in Gunn rats. Photoexposure caused a 120% increase in biliary excretion of bilirubin isomers in Gunn rats and only 40% in GT-TR- rats. This shows that the biliary excretion of bilirubin photoisomers is indeed affected by the hepatic excretion defect of GT-TR- rats and suggests that hepatic excretion of bilirubin photoisomers proceeds via the same route as other organic anions such as conjugated bilirubin and tetrabromosulphthalein.