Crigler-Najjar type I (CNI) syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by uridine diphosphoglucuronosyltransferase 1A1 (UGT1A1) deficiency. The disease is lethal due to bilirubin-induced neurological damage unless phototherapy is applied from birth. However, treatment becomes less effective during growth, and liver transplantation is required. To investigate the pathophysiology of the disease and therapeutic approaches in mice, we generated a mouse model by introducing a premature stop codon in the UGT1a1 gene, which results in an inactive enzyme. Homozygous mutant mice developed severe jaundice soon after birth and died within 11 d, showing significant cerebellar alterations. To rescue neonatal lethality, newborns were injected with a single dose of adeno-associated viral vector 9 (AAV9) expressing the human UGT1A1. Gene therapy treatment completely rescued all AAV-treated mutant mice, accompanied by lower plasma bilirubin levels and normal brain histology and motor coordination. Our mouse model of CNI reproduces genetic and phenotypic features of the human disease. We have shown, for the first time, the full recovery of the lethal effects of neonatal hyperbilirubinemia. We believe that, besides gene-addition-based therapies, our mice could represent a very useful model to develop and test novel technologies based on gene correction by homologous recombination.—Bortolussi, G., Zentilin, L., Baj, G., Giraudi, P., Bellarosa, C., Giacca, M., Tiribelli, C., Muro, A. F. Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer.
Null mutations in the UGT1A1 gene result in Crigler-Najjar syndrome type I (CNSI), characterized by severe hyperbilirubinemia and constant risk of developing neurological damage. Phototherapy treatment lowers plasma bilirubin levels, but its efficacy is limited and liver transplantation is required. To find alternative therapies, we applied AAV liver-specific gene therapy to a lethal mouse model of CNSI. We demonstrated that a single neonatal hUGT1A1 gene transfer was successful and the therapeutic effect lasted up to 17 months postinjection. The therapeutic effect was mediated by the presence of transcriptionally active double-stranded episomes. We also compared the efficacy of two different gene therapy approaches: liver versus skeletal muscle transgene expression. We observed that 5-8% of normal liver expression and activity levels were sufficient to significantly reduce bilirubin levels and maintain lifelong low plasma bilirubin concentration (3.1 -1.5 mg/dl). In contrast, skeletal muscle was not able to efficiently lower bilirubin (6.4 -2.0 mg/dl), despite 20-30% of hUgt1a1 expression levels, compared with normal liver. We propose that this remarkable difference in gene therapy efficacy could be related to the absence of the Mrp2 and Mrp3 transporters of conjugated bilirubin in muscle. Taken together, our data support the concept that liver is the best organ for efficient and long-term CNSI gene therapy, and suggest that the use of extra-hepatic tissues should be coupled to the presence of bilirubin transporters.
Programmed cell death is characterized by posttranslational modifications of a limited and specific set of nuclear proteins. We demonstrate that during apoptosis of different types of tumor cells there is a monomethylation of the nuclear protein HMGA1a that is associated to its previously described hyperphosphorylation/dephosphorylation process. HMGA1a methylation is strictly related to the execution of programmed cell death and is a massive event that involves large amounts of the protein. In some tumor cells, HMGA1a protein is already methylated to an extent that depends on cell type. The degree of methylation in any case definitely increases during apoptosis. In the studied cell systems (human leukaemia, human prostate tumor, and rat thyroid transformed cells) among the low-molecular-mass HMG proteins, only HMGA1a was found to be methylated. A tryptic digestion map of HPLC-purified HMGA1a protein showed that methylation occurs at arginine 25 in the consensus G(24)R(25)G(26) that belongs to one of the DNA-binding AT-hooks of the protein. An increase of HMGA1a methylation could be related to heterochromatin and chromatin remodeling of apoptotic cells.
Background: Severe neonatal hyperbilirubinemia, with consequent encephalopathy, remains a common cause of morbidity and death in many regions of the world. Poor access to clinical laboratory resources and screening programs to measure plasma bilirubin levels is a major contributor to delayed treatment in developing countries, and the cost of existing point-of-care screening instruments precludes their dissemination. Objectives: We are evaluating the accuracy of a low-cost, minimally invasive point-of-care system (Bilistick) requiring a 25-µl blood sample that could be used in low-resource environments to evaluate patients with neonatal jaundice. Methods: We compared plasma bilirubin levels in divided blood samples by clinical laboratories and by Bilistick at two medical centers serving term and near-term newborns from ethnically different populations. Results: 118 neonates with bilirubin levels ranging from 24.8 to 501.0 µmol/l were analyzed. The mean bilirubin concentration (±SD) was 215.6 ± 85.5 µmol/l for Bilistick and 226.1 ± 86.4 µmol/l by laboratory determination. Pearson’s correlation coefficient between all paired results was 0.961, and the Bland-Altman analysis showed a mean difference of 10.3 µmol/l with a 95% interval of agreement of –38.0 to 58.7 µmol/l. Conclusion: Bilistick is a minimally invasive method for measuring total bilirubin concentration over a wide range of values and should provide an affordable and accurate system for pre-discharge and follow-up screening of jaundiced infants, particularly in low-resource environments.
Observational epidemiological studies showed that mild hyperbilirubinemia has beneficial effects on the prevention of cardiovascular disease, type 2 diabetes mellitus, and metabolic syndrome. In mammals, bilirubin plays a major role as a potent antioxidant. Uridine 5’‐diphospho‐glucuronosyl transferase (UGT)1A1 variants coding for bilirubin UDP‐glucuronosyl transferase resulting in mild hyperbilirubinemia (as in Gilbert syndrome (GS)) may confer a strong genetic advantage. Strategies to boost bioavailability of bilirubin or to mimic GS represent an attractive approach to prevent many oxidative stress and inflammation‐mediated diseases. Even a tiny, micromolar increase in serum bilirubin concentrations substantially decreases the risk of oxidative stress–mediated diseases. There are several possible ways to achieve this, including lifestyle changes, changes in dietary patterns, regular physical activities, or use of chemical drug or of specific plant products either in the form of regular food items or nutraceuticals. Further basic and experimental research is required to fully uncover this promising therapeutic field.
ABSTRACT:Although it has been suggested that the unbound, free, (B f ) rather than total (B T ) bilirubin level correlates with cell toxicity, direct experimental evidence supporting this conclusion is limited. In addition, previous studies never included a direct measurement of B f , using newer, accurate methods. To test "the free bilirubin hypothesis", in vitro cytotoxicity was assessed in four cell lines exposed to different B f concentrations obtained by varying B T /Albumin ratio, using serum albumins with different binding affinities, and/or displacing unconjugated bilirubin (UCB) from albumin with a sulphonamide. B f was assessed by the modified, minimally diluted peroxidase method. Cytotoxicity varied among cell lines but was invariably related to B f and not B T . Light exposure decreased toxicity parallel to a decrease in B f . In the absence of albumin, no cytotoxicity was found at a B f of 150 nM whereas in the presence of albumin a similar B f resulted in a 40% reduction of viability indicating the importance of total cellular uptake of UCB in eliciting toxic effect. In the presence of albumin-bound UCB, bilirubin-induced cytotoxicity in a given cell line is accurately predicted by B f irrespective of the source and concentration of albumin, or total bilirubin level. (Pediatr Res 62: 576-580, 2007) P lasma levels of unconjugated bilirubin (UCB) are elevated in almost all newborn infants. In some infants with markedly elevated plasma UCB levels, bilirubin causes neurotoxicity, sometimes resulting in permanent neurologic dysfunction (1). Management guidelines for jaundiced term and near-term infants, published by the American Academy of Pediatrics (AAP), are based on the premise that total serum bilirubin concentration (B T ) is the best available predictor of risk for bilirubin-induced neurologic damage (BIND) (2). Clinical evidence has indicated, however, that B T , beyond a threshold value of 20 mg/dL, is a poor discriminator of individual risk for BIND (3,4). Since over 99.9% of total plasma UCB (B T ) is bound to albumin or apolipoprotein D (5) and only unbound bilirubin can enter the brain across an intact blood-brain barrier, the level of unbound "free" bilirubin (B f ) should theoretically provide a more accurate indication of the risk of kernicterus.Published studies of UCB toxicity in cell cultures, conducted with different types of albumin at varied molar ratios of bilirubin/albumin (6 -9) reported an increase in cell damage depending on B T . The hypothesis that cell injury is correlated better with B f was first suggested by Nelson et al. (10) in an in vitro study. Moreover, Ostrow et al. (11) reported a meta analysis of in vitro studies that suggested cytotoxicity was better correlated with B f (11). This conclusion was however based on B f calculations using published binding constants, rather than direct measurement in the culture medium, and was thus limited by unknown variations in binding associated with differences in the composition of the incubation media.In jaundiced newborns...
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