Megacystis Microcolon Intestinal Hypoperistalsis Syndrome: A Case Series With Long-term Follow-up and Prolonged Survival.

OBJECTIVES: Describe clinical characteristics, management, and outcome in a cohort of megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) patients. METHODS: We conducted a retrospective chart review of MMIHS patients followed at a large transplant and intestinal rehabilitation center over a period of 17 years. RESULTS: We identified 25 patients with MMIHS (68% girls, 13 transplanted). One transplanted and 1 nontransplanted patient were lost to follow-up. We estimated 100, 100, and 86% for 5-, 10-, and 20-year survival, respectively, with only 1 death. Of the 22 patients alive at the time of study (11 transplanted, 11 nontransplanted), median age was 9.2 years (range 2.7-22.9 years). Longest posttransplant follow-up was 16 years. Seventeen patients had available prenatal imaging reports; all showed distended bladder. Eight had genetic testing (5, ACTG2; 2, MYH11; 1, MYL9). Almost all patients had normal growth with median weight z-score -0.77 (interquartile range -1.39 to 0.26), height z score -1.2 (-2.04 to -0.48) and body mass index z-score 0.23 (-0.37 to 0.93) with no statistical difference between transplanted and nontransplanted patients. All nontransplanted patients were on parenteral nutrition with minimal/no feeds, and all except 1 of the transplanted patients were on full enteral feeds. Recent average bilirubin, INR, albumin, and creatinine fell within the reference ranges. CONCLUSIONS: This is the largest single-center case series with the longest duration of follow-up for MMIHS patients. In the current era of improved intestinal rehabilitation and transplantation, MMIHS patients have excellent outcomes in survival, growth, and liver function. This observation contradicts previous reports and should alter counselling and management decisions in these patients at diagnosis.

Current practices in lipid emulsion utilization in the prevention and treatment of intestinal failure-associated liver disease: A survey of pediatric intestinal rehabilitation and transplant centers.

BACKGROUND: Newer intravenous lipid emulsions (ILEs), such as fish oil-based intravenous lipid emulsions (FO-ILEs) and soybean oil, medium-chain triglycerides, olive oil, and fish oil-based intravenous lipid emulsions (SMOF-ILEs), provide alternatives to soybean oil-based intravenous lipid emulsions (SO-ILEs). We explored current ILE practice patterns among intestinal rehabilitation and transplant centers. METHODS: A survey was developed addressing ILE availability, ILE preference in clinical scenarios, and factors influencing ILE choice. This survey was reviewed locally and by the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition Intestinal Rehabilitation Special Interest Group, the Intestinal Rehabilitation and Transplant Association scientific committee, and the American Society of Parenteral and Enteral Nutrition pediatric intestinal failure section research committee. We recruited providers nationally and internationally from centers with and without intestinal transplant programs. RESULTS: We included 34 complete responses, 29 from the United States. Sixteen centers performed intestinal transplants. All centers had access to SMOF-ILEs, 85% had access to FO-ILEs, and 91% had access to SO-ILEs. In new patients, 85% use SMOF-ILEs as the first choice ILE. In those with new intestinal failure-associated liver disease (IFALD), FO-ILE was preferred to SMOF-ILE (56% vs 38%). In those developing IFALD on SMOF-ILE, 65% switched to FO-ILE, whereas 24% remained on SMOF-ILE. CONCLUSIONS: Centers have routine access to alternative ILEs, and these are quickly replacing SO-ILEs in all circumstances. Future work should focus on how this shift in practice affects outcomes to provide decision support in specific clinical scenarios.

Pollen season trends as markers of climate change impact: Betula, Quercus and Poaceae.

The incidences of respiratory allergies are at an all-time high. Pollen aeroallergens can reflect changing climate, with recent studies in Europe showing some, but not all, pollen types are increasing in severity, season duration and experiencing an earlier onset. This study aimed to identify pollen trends in the UK over the last twenty-six years for a range of pollen sites, with a focus on the key pollen types of Poaceae (grass), Betula (birch) and Quercus (oak) and to examine the relationship of these trends with meteorological factors. Betula pollen seasons show no significant trends for onset, first high day or duration but increasing pollen production in the Midlands region of the UK is being driven by warmer temperatures in the previous June and July. Quercus pollen seasons are starting earlier, due to increasing temperature and sunshine totals in April, but are not becoming more severe. The seasons are lasting longer, although no significant climate drivers for this were identified. The first high day of the Poaceae pollen season is occurring earlier in central UK regions due to an increasing trend for all temperature variables in the previous December, January, April, May and June. Severity and duration of the season show no significant trends and are spatially and temporally variable. Important changes are occurring in the UK pollen seasons that will impact on the health of respiratory allergy sufferers, with more severe Betula pollen seasons and longer Quercus pollen seasons. Most of the changes identified were caused by climate drivers of increasing temperature and sunshine total. However, Poaceae pollen seasons are neither becoming more severe nor longer. The reasons for this included a lack of change in some monthly meteorological variables, or land-use change, such as grassland being replaced by urban areas or woodland.

Valproic acid-associated acute liver failure in children: case report and analysis of liver transplantation outcomes in the United States.

OBJECTIVE: To determine whether valproic acid (VPA)-associated acute liver failure (ALF; VPA-ALF) explains the poor outcomes after liver transplantation (LT) in children. STUDY DESIGN: Organ Procurement and Transplantation Network data of pediatric patients who underwent LT for VPA-ALF and ALF caused by other drugs (non-VPA-drug-induced acute liver failure [DIALF]) were analyzed. Pre- and post-transplant variables and post-LT survival were compared between VPA-ALF and non-VPA-DIALF. RESULTS: Seventeen children were transplanted for VPA-ALF. Of the 17 children, 82% died within 1 year of LT. Pre- and post-transplant parameters of VPA versus non-VPA-DIALF were comparable with two exceptions. The median alanine aminotransferase level at transplant was remarkably lower in VPA-ALF compared with non-VPA-DIALF (45 versus 1179 IU/L, P = .004). One-year survival probability was worse in VPA-ALF than non-VPA-DIALF (20% versus 69%, P < .0001). Median post-LT survival time for VPA-ALF was 2.8 months. CONCLUSION: Children who underwent LT for VPA-ALF had a significantly lower survival probability than children with non-VPA-DIALF. Current data suggest that VPA-ALF in children represents an "unmasking" of mitochondrial disease. VPA-ALF should be a contraindication for LT, even in the absence of a documented mitochondrial disease.

Transcriptome profiling of the small intestinal epithelium in germfree versus conventional piglets.

BACKGROUND: To gain insight into host-microbe interactions in a piglet model, a functional genomics approach was used to address the working hypothesis that transcriptionally regulated genes associated with promoting epithelial barrier function are activated as a defensive response to the intestinal microbiota. Cesarean-derived germfree (GF) newborn piglets were colonized with adult swine feces, and villus and crypt epithelial cell transcriptomes from colonized and GF neonatal piglets were compared using laser-capture microdissection and high-density porcine oligonucleotide microarray technology. RESULTS: Consistent with our hypothesis, resident microbiota induced the expression of genes contributing to intestinal epithelial cell turnover, mucus biosynthesis, and priming of the immune system. Furthermore, differential expression of genes associated with antigen presentation (pan SLA class I, B2M, TAP1 and TAPBP) demonstrated that microbiota induced immune responses using a distinct regulatory mechanism common for these genes. Specifically, gene network analysis revealed that microbial colonization activated both type I (IFNAR) and type II (IFNGR) interferon receptor mediated signaling cascades leading to enhanced expression of signal transducer and activator of transcription 1 (STAT1), STAT2 and IFN regulatory factor 7 (IRF7) transcription factors and the induction of IFN-inducible genes as a reflection of intestinal epithelial inflammation. In addition, activated RNA expression of NF-kappa-B inhibitor alpha (NFkappaBIA; a.k.a I-kappa-B-alpha, IKBalpha) and toll interacting protein (TOLLIP), both inhibitors of inflammation, along with downregulated expression of the immunoregulatory transcription factor GATA binding protein-1 (GATA1) is consistent with the maintenance of intestinal homeostasis. CONCLUSION: This study supports the concept that the intestinal epithelium has evolved to maintain a physiological state of inflammation with respect to continuous microbial exposure, which serves to sustain a tight intestinal barrier while preventing overt inflammatory responses that would compromise barrier function.

Deficient and Null Variants of SERPINA1 Are Proteotoxic in a Caenorhabditis elegans Model of α1-Antitrypsin Deficiency.

α1-antitrypsin deficiency (ATD) predisposes patients to both loss-of-function (emphysema) and gain-of-function (liver cirrhosis) phenotypes depending on the type of mutation. Although the Z mutation (ATZ) is the most prevalent cause of ATD, >120 mutant alleles have been identified. In general, these mutations are classified as deficient (<20% normal plasma levels) or null (<1% normal levels) alleles. The deficient alleles, like ATZ, misfold in the ER where they accumulate as toxic monomers, oligomers and aggregates. Thus, deficient alleles may predispose to both gain- and loss-of-function phenotypes. Null variants, if translated, typically yield truncated proteins that are efficiently degraded after being transiently retained in the ER. Clinically, null alleles are only associated with the loss-of-function phenotype. We recently developed a C. elegans model of ATD in order to further elucidate the mechanisms of proteotoxicity (gain-of-function phenotype) induced by the aggregation-prone deficient allele, ATZ. The goal of this study was to use this C. elegans model to determine whether different types of deficient and null alleles, which differentially affect polymerization and secretion rates, correlated to any extent with proteotoxicity. Animals expressing the deficient alleles, Mmalton, Siiyama and S (ATS), showed overall toxicity comparable to that observed in patients. Interestingly, Siiyama expressing animals had smaller intracellular inclusions than ATZ yet appeared to have a greater negative effect on animal fitness. Surprisingly, the null mutants, although efficiently degraded, showed a relatively mild gain-of-function proteotoxic phenotype. However, since null variant proteins are degraded differently and do not appear to accumulate, their mechanism of proteotoxicity is likely to be different to that of polymerizing, deficient mutants. Taken together, these studies showed that C. elegans is an inexpensive tool to assess the proteotoxicity of different AT variants using a transgenic approach.

Acute DSS colitis alters EphB6 receptor expression in neurons of the spinal dorsal horn.

The ephrin family of receptors (Eph) and their ephrin ligands are involved in pain associated hyperalgesia, but the underlying mechanisms involved have not been fully elucidated. The EphB6 receptor is a distinctive member of the EphB subclass in that its kinase domain contains several alterations in the conserved amino acids and thus lacks catalytic activity. We sought to identify a role for EphB6 in inflammatory pain, with the murine dextran sulfate sodium (DSS) colitis model of inflammatory bowel disease (IBD). Colitis, induced with the administration of 4% (wt./vol.) DSS in the drinking water, significantly decreased EphB6 protein expression levels in neurons of the lower thoracic superficial layers of spinal dorsal horns, the location of neurons that receive the majority of nociceptive information from the colon, via the primary afferents. A shift towards increased EphB/ephrinB forward signaling, mediated by EphB6 down-regulation in neurons of the dorsal horn, may play a role in inflammatory pain caused by IBD.

Using Caenorhabditis elegans to study serpinopathies.

Protein misfolding, polymerization, and/or aggregation are hallmarks of serpinopathies and many other human genetic disorders including Alzheimer's, Huntington's, and Parkinson's disease. While higher organism models have helped shape our understanding of these diseases, simpler model systems, like Caenorhabditis elegans, offer great versatility for elucidating complex genetic mechanisms underlying these diseases. Moreover, recent advances in automated high-throughput methodologies have promoted C. elegans as a useful tool for drug discovery. In this chapter, we describe how one could model serpinopathies in C. elegans and how one could exploit this model to identify small molecule compounds that can be developed into effective therapeutic drugs.

In vitro and in vivo effects on neural crest stem cell differentiation by conditional activation of Runx1 short isoform and its effect on neuropathic pain behavior.

INTRODUCTION: Runx1, a Runt domain transcription factor, controls the differentiation of nociceptors that express the neurotrophin receptor Ret, regulates the expression of many ion channels and receptors, and controls the lamina-specific innervation pattern of nociceptive afferents in the spinal cord. Moreover, mice lacking Runx1 exhibit specific defects in thermal and neuropathic pain. We investigated whether conditional activation of Runx1 short isoform (Runx1a), which lacks a transcription activation domain, influences differentiation of neural crest stem cells (NCSCs) in vitro and in vivo during development and whether postnatal Runx1a activation affects the sensitivity to neuropathic pain. METHODS: We activated ectopic expression of Runx1a in cultured NCSCs using the Tet-ON gene regulatory system during the formation of neurospheres and analyzed the proportion of neurons and glial cells originating from NCSCs. In in vivo experiments we applied doxycycline (DOX) to pregnant mice (days 8-11), i.e. when NCSCs actively migrate, and examined the phenotype of offsprings. We also examined whether DOX-induced activation of Runx1a in adult mice affects their sensitivity to mechanical stimulation following a constriction injury of the sciatic nerve. RESULTS: Ectopic Runx1a expression in cultured NCSCs resulted in predominantly glial differentiation. Offsprings in which Runx1a had been activated showed retarded growth and displayed megacolon, pigment defects, and dystrophic dorsal root ganglia. In the neuropathic pain model, the threshold for mechanical sensitivity was markedly increased following activation of Runx1a. CONCLUSION: These data suggest that Runx1a has a specific role in NCSC development and that modulation of Runx1a activity may reduce mechanical hypersensitivity associated with neuropathic pain.

Automated high-content live animal drug screening using C. elegans expressing the aggregation prone serpin α1-antitrypsin Z.

The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.

Paraneoplastic anti-N-methyl-D-aspartate receptor encephalitis associated with ovarian teratoma.

OBJECTIVE: To report the autoantigens of a new category of treatment-responsive paraneoplastic encephalitis. METHODS: Analysis of clinical features, neuropathological findings, tumors, and serum/cerebrospinal fluid antibodies using rat tissue, neuronal cultures, and HEK293 cells expressing subunits of the N-methyl-D-aspartate receptor (NMDAR). RESULTS: Twelve women (14-44 years) developed prominent psychiatric symptoms, amnesia, seizures, frequent dyskinesias, autonomic dysfunction, and decreased level of consciousness often requiring ventilatory support. All had serum/cerebrospinal fluid antibodies that predominantly immunolabeled the neuropil of hippocampus/forebrain, in particular the cell surface of hippocampal neurons, and reacted with NR2B (and to a lesser extent NR2A) subunits of the NMDAR. NR2B binds glutamate and forms heteromers (NR1/NR2B or NR1/NR2A/NR2B) that are preferentially expressed in the adult hippocampus/forebrain. Expression of functional heteromers (not single subunits) was required for antibody binding. Eleven patients had teratoma of the ovary (six mature) and one a mature teratoma in the mediastinum; five of five tumors examined contained nervous tissue that strongly expressed NR2 subunits and reacted with patients' antibodies. Tumor resection and immunotherapy resulted in improvement or full recovery of eight of nine patients (paralleled by decreased antibody titers); two of three patients without tumor resection died of neurological deterioration. Autopsies showed extensive microgliosis, rare T-cell infiltrates, and neuronal degeneration predominantly involving, but not restricted to, the hippocampus. INTERPRETATION: Antibodies to NR2B- and NR2A-containing heteromers of the NMDAR associate with a severe but treatment-responsive encephalitis. Our findings provide a diagnostic test and suggest a model of autoimmune NMDAR-related encephalitis with broad implications for other immune-mediated disorders of memory, behavior, and cognition.

An asymmetric contribution to gamma-aminobutyric type A receptor function of a conserved lysine within TM2-3 of alpha1, beta2, and gamma2 subunits.

Mutations that impair the expression and/or function of gamma-aminobutyric acid type A (GABAA) receptors can lead to epilepsy. The familial epilepsy gamma2(K289M) mutation affects a basic residue conserved in the TM2-3 linker of most GABAA subunits. We investigated the effect on expression and function of the Lys --> Met mutation in mouse alpha1(K278M), beta2(K274M), and gamma2(K289M) subunits. Compared with cells expressing wild-type and alpha1beta2gamma2(K289M) receptors, cells expressing alpha1(K278M)beta2gamma2 and alpha1beta2(K274M)gamma2 receptors exhibited reduced agonist-evoked current density and reduced GABA potency, with no change in single channel conductance. The low current density of alpha1beta2(K274M)gamma2 receptors coincided with reduced surface expression. By contrast the surface expression of alpha1(K278M)beta2gamma2 receptors was similar to wild-type and alpha1beta2gamma2(K289M) receptors suggesting that the alpha1(K278M) impairs function. In keeping with this interpretation GABA-activated channels mediated by alpha1(K278M)beta2gamma2 receptors had brief open times. To a lesser extent gamma2(K289M) also reduced mean open time, whereas beta2(K274M) had no effect. We used propofol as an alternative GABAA receptor agonist to test whether the functional deficits of mutant subunits were specific to GABA activation. Propofol was less potent as an activator of alpha1(K278M)beta2gamma2 receptors. By contrast, neither beta2(K274M) nor gamma2(K289M) affected the potency of propofol. The beta2(K274M) construct was unique in that it reduced the efficacy of propofol activation relative to GABA. These data suggest that the alpha1 subunit Lys-278 residue plays a pivotal role in channel gating that is not dependent on occupancy of the GABA binding site. Moreover, the conserved TM2-3 loop lysine has an asymmetric function in different GABAA subunits.

The epilepsy mutation, gamma2(R43Q) disrupts a highly conserved inter-subunit contact site, perturbing the biogenesis of GABAA receptors.

Given the association of a gamma2 mutation (R43Q) with epilepsy and the reduced cell surface expression of mutant receptors, we investigated a role for this residue in alpha1beta2gamma2 receptor assembly when present in each subunit. Regardless of which subunit contained the mutation, mutant GABA(A) receptors assembled poorly into functional cell surface receptors. The low level of functional expression gives rise to reduced GABA EC50s (alpha1(R43Q)beta2gamma2 and alpha1beta2(R43Q)gamma2) or reduced benzodiazepine potentiation of GABA-evoked currents (alpha1beta2gamma2(R43Q)). We determined that a 15-residue peptide surrounding R43 is capable of subunit binding, with a profile that reflected the orientation of subunits in the pentameric receptor. Subunit binding is perturbed when the R43Q mutation is present suggesting that this residue is critical for the formation of inter-subunit contacts at (+) interfaces of GABAA subunits. Rather than being excluded from receptors, gamma2(R43Q) may form non-productive subunit interactions leading to a dominant negative effect on other receptor subtypes.

Postnatal ontogeny of kinetics of porcine jejunal brush border membrane-bound alkaline phosphatase, aminopeptidase N and sucrase activities.

Our objectives were to determine postnatal changes in the maximal enzyme activity (V(max)) and enzyme affinity (K(m)) of jejunal mucosal membrane-bound alkaline phosphatase, aminopeptidase N and sucrase using a porcine model which may more closely resemble the human intestine. Jejunal brush border membrane was prepared by Mg(2+)-precipitation and differential centrifugation from pigs of suckling (8 days), weaning (28 days), post-weaning (35 days) and adult (70 days) stages. p-Nitrophenyl phosphate (0-8 mM), L-alanine-p-nitroanilide hydrochloride (0-28 mM) and sucrose (0-100 mM) were used in alkaline phosphatase, aminopeptidase N and sucrase kinetic measurements. V(max) of alkaline phosphatase was the lowest in the adult (4.27 micromol.mg(-1) protein.min(-1)), intermediate in the suckling (9.75 micromol.mg(-l) protein.min(-l)) and the highest in the weaning and post-weaning stage (12.83 and 10.40 micromol.mg(-l) protein.min(-l)). K(m) of alkaline phosphatase was high in the suckling and weaning stages (5.14 and 9.93 mM) and low in the adult (0.66 mM). V(max) of aminopeptidase N was low in the suckling (7.04 micromol.mg protein(-1).min(-1)) and high in the post-weaning stage (13.36 micromol.mg(-l) protein.min(-l)). K(m) of aminopeptidase N was the highest in the two weaning stages (2.96 and 3.39 mM), intermediate in the adult (2.33 mM) and the lowest in the suckling stage (1.66 mM). V(max) of sucrase increased from the suckling to the adult (0.48-1.30 micromol.mg(-l) protein.min(-l)). K(m) of sucrase ranged from 11.19 to 16.57 mM. There are dramatic postnatal developmental changes in both the maximal enzyme activity and enzyme affinity of jejunal brush border membrane-bound alkaline phosphatase, aminopeptidase N and sucrase in the pig.

GABA(A) receptor composition is determined by distinct assembly signals within alpha and beta subunits.

Key to understanding how receptor diversity is achieved and controlled is the identification of selective assembly signals capable of distinguishing between other subunit partners. We have identified that the beta1-3 subunits exhibit distinct assembly capabilities with the gamma2L subunit. Similarly, analysis of an assembly box in alpha1-(57-68) has revealed an absolute requirement for this region in the assembly of alphabeta receptors. Furthermore, a selective requirement for a single amino acid (Arg-66), previously shown to be essential for the formation of the low affinity GABA binding site, is observed. This residue is critical for the assembly of alpha1beta2 but not alpha1beta1 or alpha1beta3 receptors. We have confirmed the ability of the previously identified GKER signal in beta3 to direct the assembly of betagamma receptors. The GKER signal is also involved in driving assembly with the alpha1 subunit, conferring the ability to assemble with alpha1(R66A) on the beta2 subunit. Although this signal is sufficient to permit the formation of beta2gamma2 receptors, it is not necessary for beta3gamma2 receptor formation, suggesting the existence of alternative assembly signals. These findings support the belief that GABA(A) receptor assembly occurs via defined pathways to limit the receptor diversity.

Measurement of erythrocyte C4d and complement receptor 1 in systemic lupus erythematosus.

OBJECTIVE: C4-derived activation fragments are the only complement ligands present on the surfaces of normal erythrocytes. The significance of this observation is unknown, and the role of erythrocyte-bound C4 (E-C4) in human disease has not been explored. More than any other human disease, the pathogenesis of systemic lupus erythematosus (SLE) has been characterized by defects in clearance of complement-bearing immune complexes via erythrocytes expressing complement receptor 1 (CR1). This study was undertaken to determine whether these functional defects might be reflected by abnormal patterns of E-C4 and E-CR1 expression on erythrocytes of patients with SLE. METHODS: We conducted a cross-sectional study of 100 patients with SLE, 133 patients with other diseases, and 84 healthy controls. Erythrocytes were characterized by indirect immunofluorescence and by flow cytometry for determination of levels of C4d and CR1. RESULTS: Patients with SLE had higher levels of E-C4d and lower levels of E-CR1 than did patients with other diseases (P < or = 0.001) or healthy controls (P < or = 0.001). The test was 81% sensitive and 91% specific for SLE versus healthy controls and 72% sensitive and 79% specific for SLE versus other diseases, and it had an overall negative predictive value of 92%. CONCLUSION: This is the first report of abnormal levels of E-C4d in human disease. We found that abnormally high levels of E-C4d and low levels of E-CR1 are characteristic of SLE, and combined measurement of the 2 molecules has high diagnostic sensitivity and specificity for lupus. Determination of E-C4d/E-CR1 levels may be a useful addition to current tests and criteria for SLE diagnosis.