A non-immunogenic adenoviral vector, coexpressing CTLA4Ig and bilirubin-uridine-diphosphoglucuronateglucuronosyltransferase permits long-term, repeatable transgene expression in the Gunn rat model of Crigler-Najjar syndrome.
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Host immune responses limit the duration of expression of transgenes introduced by recombinant adenoviruses, preclude gene transfer upon vector readministration and cause liver injury. CTLA4Ig inhibits immune response by blocking the co-stimulatory interaction between CD28 on T cells and B7 on antigen-presenting cells. We have constructed a recombinant adenovirus, Ad-hUGT1A1-CTLA4Ig that coexpresses human bilirubin-uridinediphosphoglucuronate glucuronosyltransferase (hUGT1A1) and soluble murine CTLA4Ig, both driven by CMV immediate-early promoters. After intravenous injection of this vector (6 x 10(11) p.f.u.) into UGT1A1-deficient jaundiced Gunn rats, serum CTLA4Ig levels peaked at 1.8-2.0 mg/ml on day 7 and declined thereafter to 0.2 mg/ml by day 180. Serum bilirubin declined from mean preinjection levels of 8.0 mg/dl to 0.48-0.6 mg/dl in 3 days, remained normal for 28 weeks, and then gradually increased to 8 mg/dl by day 350. A second injection of Ad-hUGT1A1-CTLA4Ig normalized serum bilirubin. In two rats in this group that were followed longer, serum bilirubin increased to 3.1 and 3.5 mg/dl in 40 weeks, but was normalized again after a third injection. The antibody and cytotoxic lymphocyte (CTL) responses were negligible, and liver biopsy showed no inflammatory cell infiltration. Rats receiving a tertiary challenge with Ad-LacZ (expressing E. coli beta-galactosidase) (5 x 10(11) p.f.u.), 2 months after the second dose of Ad-hUGT1A1-CTLA4Ig, showed beta-galactosidase expression in over 80% of hepatocytes. In contrast, after Ad-hUGT1A1 (which expresses UGT1A1 alone) injection, serum bilirubin remained normal for only 4 weeks, and returned to preinjection levels by day 120. Bilirubin levels did not decline upon reinjection, and beta-galactosidase was not expressed after Ad-LacZ. High levels of adenovirus-specific antibodies and CTL, and hepatic inflammation were found. This is the first demonstration that coexpression of CTLA4Ig permits prolonged expression and repeatable gene transfer by an adenoviral vector. (+info)
In vitro effects of light on serum bilirubin subfractions measured by high-performance liquid chromatography: comparison with four routine methods.
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The effects of light on serum bilirubin subfractions in vitro were investigated by HPLC and four routine methods for bilirubin analysis. By HPLC, the rate of photodegradation of unconjugated bilirubin (Bu) was nearly twice that of monoconjugated bilirubin (mBc) and threefold that of diconjugated bilirubin (dBc); delta bilirubin (Bd) was most stable against photoirradiation. In the diazo method, the rate of photodegradation of direct bilirubin was almost the same as that of the sum of mBc, dBc, and Bd determined by the HPLC method. However, the rate of photodegradation of indirect bilirubin was significantly lower (P < 0.001) than that obtained by HPLC, because approximately 30% of the bilirubin photoproducts reacted with the diazo reagent as indirect bilirubin. The rate of photodegradation of total bilirubin determined by the direct spectrometric method was lower than that determined by the diazo method, but equal to that of the total peak areas of HPLC. In the Ektachem method, bilirubin photoproducts affected total bilirubin negligibly, and Bc and Bu positively, so that the value of Bd decreased. In the bilirubin oxidase method, bilirubin photoproducts were oxidized enzymatically by both the total and direct bilirubin reagents. We re-emphasize the importance of shielding serum from light to avoid generating bilirubin photoproducts that interfere with the accurate determination of serum bilirubin subfractions. We also recommend HPLC analysis as a standard method for bilirubin measurement. (+info)
Permanent access to the portal system for cellular transplantation using an implantable port device.
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A novel application of the implantable Port-a-Cath (PAC) system is described in the context of cellular transplantation. A silicone catheter was inserted in a collateral branch of the portal vein and connected to a port device positioned subcutaneously on the left thoracic cage. This permanent vascular access allowed iterative intraportal infusions of allogenic hepatocytes without the need of repeated transhepatic catheterization of the portal vein. Using this technique, repeated infusions of cryopreserved and / or fresh hepatocytes were successfully carried out in 3 children with inborn errors of liver metabolism, with the aim of progressively providing a sufficient mass of transplanted liver cells to stabilize the metabolic condition of the patients. We suggest that this technique might also be valuable in pancreatic islet cell transplantation. (+info)
Paternal isodisomy for chromosome 2 as the cause of Crigler-Najjar type I syndrome.
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Crigler-Najjar syndrome type I (CN-I) is a rare and severe autosomal recessive metabolic disease due to a total deficiency of bilirubin uridine diphosphate glucuronosyltransferase located on chromosome 2. We report on a child with CN-I due to a phenylalanine residue deletion inherited only from the father carrying this deletion at the heterozygous state. Cytogenetic analyses showed no deletion of the chromosomal 2q37 region. Microsatellite analysis of the child and his parents was consistent with paternal isodisomy for chromosome 2 in the child. This report demonstrates that uniparental disomy may be at the origin of very rare diseases transmitted as autosomal recessive traits and emphasizes the need for parental DNA analysis in such cases. (+info)
Overcoming the portal steal phenomenon in auxiliary partial orthotopic liver transplantation by modulation of the venous outflow of the native liver.
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The main drawback of auxiliary partial orthotopic liver transplantation (APOLT) is the competition of the portal flow between the graft and the native liver, leading to graft failure. In two patients with Crigler-Najjar syndrome type I, the intrahepatic resistance of the native liver was increased by occluding the recipients middle hepatic vein during parenchymal transection, leading the portal flow towards the graft. This new surgical technique could encourage centers to recommence APOLT. (+info)
Mechanisms of inherited deficiencies of multiple UDP-glucuronosyltransferase isoforms in two patients with Crigler-Najjar syndrome, type I.
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Crigler-Najjar syndrome, type I (CN-I) is a potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from a recessively inherited deficiency of hepatic UDP-glucuronosyl-transferase (UGT) activity toward bilirubin (B-UGT). Two forms of B-UGT exist in human liver. mRNAs for these two forms and that for another isoform with activity toward simple phenols (P-UGT) have unique 5' regions, but their 3' regions are identical. The three mRNA species are derived from a single locus; the unique 5' regions are encoded by single unique exons and the identical 3' regions consist of four consecutive exons that are shared by all three isoforms. In this paper, we determined genetic lesions in two CN-I patients with deficiency of hepatic B-UGT and P-UGT activities. In one patient, there was a C----T substitution in exon 4 (common region) predicting the substitution of a serine residue with a phenylalanine residue; this mutation was present in the identical region of B-UGT and P-UGT mRNAs. In the other patient, a C----T substitution in exon 2 (common region) of the B-UGT/P-UGT locus resulted in a premature stop codon. This exon (132 nt) was absent in heptic B-UGT and P-UGT mRNAs of this patient due to exon skipping during pre-mRNA processing. Sequence abnormality of three distinct mRNA species explains the abnormality of multiple UGT isoforms in these patients. Presence of identical abnormalities in the common regions of the three mRNAs is consistent with the finding that the common 3' regions of the two B-UGT mRNAs and the P-UGT mRNA are encoded by four shared exons. (+info)
Identification of a genetic alteration in the code for bilirubin UDP-glucuronosyltransferase in the UGT1 gene complex of a Crigler-Najjar type I patient.
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Patients with Crigler-Najjar syndrome (CN) type I inherit an autosomal recessive trait for hyperbilirubinemia, which is characterized by the total absence of bilirubin UDP-glucuronosyltransferase (transferase) activity. The recent identification of two bilirubin transferase isoforms with identical carboxyl termini (Ritter, J. K., J. M. Crawford, and I. S. Owens. 1991. J. Biol. Chem. 266:1043-1047) led to the discovery of a unique locus, UGT1, which encodes a family of UDP-glucuronosyltransferase isozymes, including the two bilirubin forms (Ritter, J. K., F. Chen, Y. Y. Sheen, H. M. Tran, S. Kimura, M. T. Yeatman, and I. S. Owens. 1992. J. Biol. Chem. 267:3257-3261). The UGT1 locus features a complex of six overlapping transcriptional units encoding transferases, each of which shares the four most 3' exons (2, 3, 4, and 5) specifying the 3' half of the transferase coding regions (condons 289-533) and the entire 3' untranslated region of each mRNA. This gene model predicts that a single critical mutation in any of these four "common" exons may inactivate the entire family of encoded transferases. In agreement with this prediction, we show here that in the first CN type I individual analyzed (patient F.B.), a 13-bp deletion has occurred in exon 2. Analysis of product generated by the polymerase chain reaction and genomic DNA demonstrated that F.B. is homozygous for the defective allele (UGT1*FB), and that the consanguineous parents are both heterozygotic at this locus. The mutation is predicted to result in the synthesis of severely truncated bilirubin transferase isozymes that are lacking a highly conserved sequence in the carboxyl-terminus and the characteristic membrane (endoplasmic reticulum)-anchoring segment of the protein molecule. (+info)
Seven novel mutations of the UGT1A1 gene in patients with unconjugated hyperbilirubinemia.
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The aim of this study was to identify new pathogenic variations of the UGT1A1 gene in 11 patients diagnosed with neonatal unconjugated hyperbilirubinemia. We describe two cases in which clinically unapparent heterozygotic mutations in the UGT1A1 gene may become evident in combination with certain environmental conditions or additional genetic defects. (+info)