Jon F. Watchko

Hyperbilirubinemia in the Newborn Infant 35 Weeks' Gestation: An Update With Clarifications

In July 2004, the Subcommittee on Hyperbilirubinemia of the American Academy of Pediatrics (AAP) published its clinical practice guideline on the management of hyperbilirubinemia in the newborn infant ≥35 weeks of gestation,1 and a similar guideline was published in 2007 by the Canadian Paediatric Society.2 Experience with implementation of the AAP guideline suggests that some areas require clarification. The 2004 AAP guideline also expressed hope that its implementation would “reduce the incidence of severe hyperbilirubinemia and bilirubin encephalopathy… .” We do not know how many practitioners are following the guideline, nor do we know the current incidence of bilirubin encephalopathy in the United States. We do know, however, that kernicterus is still occurring in the United States, Canada, and Western Europe.3–7 In 2002, the National Quality Forum suggested that kernicterus should be classified as a “serious reportable event,”8 sometimes termed a “never event,”9 implying that with appropriate monitoring, surveillance, and intervention, this devastating condition can, or should, be eliminated. Although this is certainly a desirable objective, it is highly unlikely that it can be achieved given our current state of knowledge and practice.10 In certain circumstances (notably, glucose-6-phosphate dehydrogenase [G6PD] deficiency, sepsis, genetic predisposition, or other unknown stressors), acute, severe hyperbilirubinemia can occur and can produce brain damage despite appropriate monitoring and intervention. In addition to clarifying certain items in the 2004 AAP guideline, we recommend universal predischarge bilirubin screening using total serum bilirubin (TSB) or transcutaneous bilirubin (TcB) measurements, which help to assess the risk of subsequent severe hyperbilirubinemia. We also recommend a more structured approach to management and follow-up according to the predischarge TSB/TcB, gestational age, and other risk factors for hyperbilirubinemia. These recommendations represent a consensus of expert opinion based on the available evidence, and they are supported by … Address correspondence to M. Jeffrey Maisels, MB, BCh, DSc, Beaumont Childrens Hospital, 3601 W. 13 Mile Rd, Royal Oak, MI 48073. E-mail: JMaisels{at}beaumont.edu

Adeno-associated virus vector-mediated minidystrophin gene therapy improves dystrophic muscle contractile function in mdx mice.

Duchenne muscular dystrophy (DMD) is the most common disabling and lethal genetic muscle disorder, afflicting 1 of every 3500 males. Patients with DMD experience progressive muscle degeneration and weakness and succumb to respiratory or cardiac failure by their early twenties. No treatment is currently available for DMD. Mutations in the dystrophin gene result in lack of a functional dystrophin protein in striated muscle, which induces instability in the muscle cell membrane leading to persistent muscle injury after contraction. We have previously created novel minidystrophin genes and demonstrated that adeno-associated virus (AAV)-mediated intramuscular delivery of the minigenes effectively ameliorated mdx dystrophic histopathology and led to normal cell membrane integrity for more than 1 year. In this paper, we investigated whether AAV-minidystrophin could also improve mdx muscle contractile function. Two-month-old adult male mdx mice, with established muscular dystrophy, were given a single-dose injection of an AAV-minidystrophin vector in the tibialis anterior (TA) muscle of one leg, with the untreated contralateral leg used as a control. The treated TA muscle showed both (1) a significant increase in isometric force generation and (2) a significant increase in resistance to lengthening activation-induced muscle force decrements. We conclude that AAV-minidystrophin gene treatment is effective in improving mdx muscle contractile function.

Bilirubin-Induced Neurologic Damage — Mechanisms and Management Approaches

The complex cascade of molecular and cellular events leading to bilirubin-induced neurotoxicity remains incompletely delineated. This review discusses bilirubin-induced brain damage and recent insights into its pathogenesis and prevention.

ABC Transporter (P-gp/ABCB1, MRP1/ABCC1, BCRP/ABCG2) Expression in the Developing Human CNS

P-glycoprotein (P-gp/ABCB1), multidrug resistance associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) are plasma membrane efflux pumps that limit the intracellular uptake and retention of numerous lipophilic, amphipathic xeno- and endobiotics. Little is known about the neonatal and developmental expression of P-gp/ABCB1, MRP1/ABCC1, and BCRP/ABCG2 in the human central nervous system (CNS), therefore post-mortem CNS tissue from infants born at 22 (0/7)-42 (0/7) weeks of gestation and adults was immunostained to determine their ontogeny and cellular localization. P-gp/ABCB1 immunostaining was observed in microvessel endothelial cells as early as 22 (0/7) weeks, increasing in prevalence and intensity with maturation, and later in gestation in large pyramidal neurons. MRP1/ABCC1 immunostaining was prominent early in the choroid plexus and ventricular ependyma, and noted later in large pyramidal neurons. BCRP/ABCG2 expression was limited to microvessel endothelial cells. P-gp/ABCB1, MRP1/ABCC1 and BCRP/ABCG2 in adult brain matched term newborn CNS but with more intense immunostaining. We conclude that P-gp/ABCB1, MRP1/ABCC1, and BCRP/ABCG2 are expressed in a developmental, cell specific, fashion in the human CNS. The complementary pattern of P-gp/ABCB1 and BCRP/ABCG2 at the blood-brain with MRP1/ABCC1 at the blood-CSF barriers may limit CNS uptake and retention of drugs and toxins in neonates.

Full Functional Rescue of a Complete Muscle (TA) in Dystrophic Hamsters by Adeno-Associated Virus Vector-Directed Gene Therapy

ABSTRACT Limb girdle muscular dystrophy (LGMD) 2F is caused by mutations in the δ-sarcoglycan (SG) gene. Previously, we have shown successful application of a recombinant adeno-associated virus (AAV) vector for genetic and biochemical rescue in the Bio14.6 hamster, a homologous animal model for LGMD 2F (J. Li et al., Gene Ther. 6:74–82, 1999). In this report, we show efficient and long-term δ-SG expression accompanied by nearly complete recovery of physiological function deficits after a single-dose AAV vector injection into the tibialis anterior muscle of the dystrophic hamsters. AAV vector treatment led to more than 97% recovery in muscle strength for both the specific twitch force and the specific tetanic force, when compared to the age-matched control. Vector treatment also prevented pathological muscle hypertrophy and resulted in normal muscle weight and size. Finally, vector-treated muscle showed substantial improvement of the histopathology. This is the first report of successful functional rescue of an entire muscle after AAV-mediated gene delivery. This report also demonstrates the feasibility of in vivo gene therapy for LGMD patients by using AAV vectors.

Kernicterus and the molecular mechanisms of bilirubin-induced CNS injury in newborns.

Kernicterus is a devastating, chronic disabling neurological disorder whose central nervous system (CNS) sequelae reflect both a predilection of bilirubin toxicity for neurons (rather than glial cells) and the regional topography of bilirubin-induced neuronal injury that is characterized by prominent basal ganglia, cochlear, and oculomotor nuclei involvement. The molecular pathogenesis of bilirubin-induced neuronal cell injury, although incompletely understood, likely reflects the untoward effects of hazardous unconjugated bilirubin concentrations on plasma, mitochondrial, and/or endoplasmic reticulum (ER) membranes. These membrane perturbations, in turn, might lead to the genesis of neuronal excitotoxicity, mitochondrial energy failure, or increased intracellular calcium concentration [Ca2+]i. These three phenomena are likely to be linked spatially and temporally in the pathogenesis of bilirubininduced neuronal injury.Downstream events triggered by increased [Ca2+]i may include, among others, the activation of proteolytic enzymes, apoptotic pathways, and/or necrosis, the individual occurrence of which is likely a function of the degree and duration of bilirubin exposure. A recent study demonstrates the activation of mitogen-activated protein kinase signal transduction pathways by bilirubin heralding a degree of complexity regarding the molecular mechanism(s) of bilirubin-induced neurotoxicity not previously appreciated. There remains, however, a paucity of data regarding specific effects of bilirubin on intracellular signaling and cell death pathways, particularly in vivo. An enhanced understanding of the molecular pathogenesis of bilirubin-induced neuronal injury will lead to the identification of potential novel interventional strategies to protect the CNS against kernicterus.

An approach to the management of hyperbilirubinemia in the preterm infant less than 35 weeks of gestation.

We provide an approach to the use of phototherapy and exchange transfusion in the management of hyperbilirubinemia in preterm infants of <35 weeks of gestation. Because there are limited data for evidence-based recommendations, these recommendations are, of necessity, consensus-based. The recommended treatment levels are based on operational thresholds for bilirubin levels and represent those levels beyond which it is assumed that treatment will likely do more good than harm. Long-term follow-up of a large population will be needed to evaluate whether or not these recommendations should be modified.

Brain Bilirubin Content Is Increased in P-Glycoprotein-Deficient Transgenic Null Mutant Mice

P-glycoprotein (P-gp), encoded by the mdr1a gene, is an ATP-dependent plasma membrane protein that is expressed in abundance on the blood-brain barrier (BBB). P-gp limits the CNS influx and retention of a variety of lipophilic compounds. We hypothesized that brain bilirubin content after an i.v. bilirubin infusion would be increased in P-gp-deficient mdr1a null mutant transgenic mice (mdr1a(-/-)) compared with controls. Eighteen mdr1a(-/-) null mutant and 18 P-gp-sufficient wild type mice (+/+) were anesthetized and 50 mg/kg bilirubin infused through the tail vein. Brain bilirubin content (mean ± SEM) 10 min after infusion was significantly higher in mdr1a(-/-) (18.1 ± 2.4 nmol/g) compared with (+/+) mice (10.4 ± 1.0 nmol/g). Brain bilirubin content declined 60 min after infusion but remained higher in mdr1a(-/-) (10.3 ± 1.4 nmol/g) compared with (+/+) mice (5.3 ± 0.9 nmol/g). Brain bilirubin clearance did not differ between groups (t1/2 ∼ 55 min). We conclude that P-gp-deficient mdr1a(-/-) mice have significantly higher brain bilirubin content compared with controls after an i.v. bilirubin load. These data suggest that 1) bilirubin is a substrate for P-gp and 2) the increased brain bilirubin content in mdr1a(-/-) mice is due to enhanced brain bilirubin influx. We speculate that BBB P-gp provides a protective effect against bilirubin neurotoxicity by reducing brain bilirubin influx.

P-Glycoprotein Expression in Mouse Brain Increases with Maturation

The mdr1a isoform of P-glycoprotein (Pgp) is an integral plasma membrane efflux pump expressed in adult brain capillary endothelial cells and astrocytes of the blood-brain barrier. We determined the developmental pattern of Pgp expression in brain tissue at embryonic day 16 (E16), day of life 0 (D0), day of life 7 (D7), day of life 21 (D21), and adults (Ad). The relative expression of Pgp mRNA and protein was indexed as a percent (mean ± SEM) of D0 levels. Pgp mRNA levels increased significantly (p < 0.01) with maturation (E16: 75 ± 8%; D21: 303 ± 37%, and Ad: 1,160 ± 120%). Similarly, Pgp protein expression was observed at E16 and increased significantly (p < 0.01) during development (E16: 52 ± 8%; D7: 187 ± 23%; D21: 440 ± 48%, and Ad: 441 ± 56%). This developmental pattern of enhanced blood-brain barrier Pgp expression with maturation was confirmed by immunohistochemistry. We conclude that (i) Pgp expression in mouse brain is limited during late embryogenesis and the newborn period; (ii) Pgp expression increases markedly with postnatal maturation, and (iii) by D21 brain Pgp protein expression approximates adult levels.

Effect of Injecting Primary Myoblasts Versus Putative Muscle-Derived Stem Cells on Mass and Force Generation in mdx Mice

It is well established that the injection of normal myoblasts or of muscle-derived stem cells (MDSCs) into the muscle of dystrophin-deficient mdx mice results in the incorporation of a number of donor myoblasts into the host muscle. However, the effect of the injected exogenous cells on mdx muscle mass and functional capacity has not been evaluated. This study evaluates the mass and functional capacity of the extensor digitorum longus (EDL) muscles of adult, male mdx mice that received intramuscular injections of primary myoblasts or of MDSCs (isolated by a preplating technique; Qu, Z., Balkir, L., van Deutekom, J.C., Robbins, P.D., Pruchnic, R., and Huard, J., J. Cell Biol. 1998;142:1257-1267) derived from normal mice. Evaluations were made 9 weeks after cell transplantation. Uninjected mdx EDL muscles have a mass 50% greater than that of age-matched C57BL/10J (normal) EDL muscles. Injections of either primary myoblasts or MDSCs have no effect on the mass of mdx EDL muscles. EDL muscles of mdx mice generate 43% more absolute twitch tension and 43% less specific tetanic tension then do EDL muscles of C57BL/10J mice. However, the absolute tetanic and specific twitch tension of mdx and C57BL/10J EDL muscles are similar. Injection of either primary myoblasts or MDSCs has no effect on the absolute or specific twitch and tetanic tensions of mdx muscle. Approximately 25% of the myofibers in mdx EDL muscles that received primary myoblasts react positively with antibody to dystrophin. There is no significant difference in the number of dystrophin-positive myofibers when MDSCs are injected. Regardless of the source of donor cells, dystrophin is limited to short distances (60-900 microm) along the length of the myofibers. This may, in part, explain the failure of cellular therapy to alter the contractile properties of murine dystrophic muscle.