Nikhil Thapar

Advances in ontogeny of the enteric nervous system

Abstractu2002 The neurons and glia that comprise the enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract, are derived from vagal and sacral regions of the neural crest. In order to form the ENS, neural crest‐derived precursors undergo a number of processes including survival, migration and proliferation, prior to differentiation into neuronal subtypes, some of which form functional connections with the gut smooth muscle. Investigation of the developmental processes that underlie ENS formation has progressed dramatically in recent years, in no small part due to the attention of scientists from a range of disciplines on the genesis of Hirschsprungs disease (aganglionic megacolon), the major congenital abnormality of the ENS. This review summarizes recent advances in the field of early ENS ontogeny and focuses on: (i) the spatiotemporal migratory pathways followed by vagal and sacral neural crest‐derived ENS precursors, including recent in vivo imaging of migrating crest cells within the gut, (ii) the roles of the RET and EDNRB signalling pathways and how these pathways interact to control ENS development, and (iii) how perpendicular migrations of neural crest cells within the gut lead to the formation of the myenteric and submucosal plexi located between the smooth muscle layers of the gut wall.

High-resolution colonic manometry accurately predicts colonic neuromuscular pathological phenotype in pediatric slow transit constipation.

Backgroundu2002 Severe pediatric slow transit constipation (STC) is commonly due to intrinsic colonic neuromuscular disease. We sought to correlate neuromuscular histological phenotypes in pediatric STC with colonic manometric phenotypes using high‐resolution manometry (HRM). We tested the hypothesis that failure of motor quiescence (FQ) between bisacodyl‐induced high amplitude propagating sequences (HAPSs) might predict neuromuscular pathology.

Pediatric Esophageal High-Resolution Manometry: Utility of a Standardized Protocol and Size-Adjusted Pressure Topography Parameters

OBJECTIVES:Esophageal high-resolution manometry (EHRM) has evolved rapidly from a research tool to a routine investigation in adult clinical practice. This study proposes and evaluates a standardized EHRM protocol for use in pediatric clinical practice.METHODS:Thirty pediatric patients underwent unsedated EHRM. Indications for EHRM were dysphagia, feeding difficulty, or pre-fundoplication assessment. Two 20-channel customized water-perfused silicone catheters, with an outside diameter of 3.8u2009mm (MuiScientific, Ontario, CA), were used. The catheters had one distal gastric channel, five channels 0.5u2009cm apart for the e-sleeve, and 14 proximal channels either 1u2009cm (for children <5 years) or 2u2009cm apart (for children >5 years). Single wet swallows, multiple rapid swallows (MRS), and solid swallows were systematically studied.RESULTS:The median age was 10 years (range 6 months–15 years). The esophageal motor findings were normal peristalsis (n=15), peristaltic dysfunction (n=12), achalasia (n=3), and spasm on consumption of solid food (n=2). The distal contractile integral adjusted for esophageal length (DCIa) of patients with peristaltic dysfunction was significantly lower than that of patients without peristaltic dysfunction (P<0.001). On MRS, aperistalsis with lack of esophagogastric junction (EGJ) relaxation was observed in patients with achalasia, and aperistalsis with complete EGJ relaxation was observed in patients with severe peristaltic dysfunction. On consumption of solid food, esophageal spasm associated with bolus impaction was observed in two patients.CONCLUSIONS:This study provides objective information with regard to topography pressure parameters in esophageal motility disorders of childhood while using a standardized EHRM protocol. The new DCIa variable may be useful for the assessment of patients with peristaltic dysfunction.

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies

Over the last 20 years, there has been increasing focus on the development of novel stem cell based therapies for the treatment of disorders and diseases affecting the enteric nervous system (ENS) of the gastrointestinal tract (so-called enteric neuropathies). Here, the idea is that ENS progenitor/stem cells could be transplanted into the gut wall to replace the damaged or absent neurons and glia of the ENS. This White Paper sets out experts views on the commonly used methods and approaches to identify, isolate, purify, expand and optimize ENS stem cells, transplant them into the bowel, and assess transplant success, including restoration of gut function. We also highlight obstacles that must be overcome in order to progress from successful preclinical studies in animal models to ENS stem cell therapies in the clinic.

Variants in RET Associated With Hirschsprung's Disease Affect Binding of Transcription Factors and Gene Expression

BACKGROUND & AIMSnTwo noncoding variations in RET-the T allele of the single nucleotide polymorphism (SNP) rs2435357 (Enh1:C>T) and the A allele of the SNP rs2506004 (Enh2:C>A)-are associated with Hirschsprungs disease. These SNPs are in strong linkage disequilibrium and located in an enhancer element in intron 1 of the RET gene. The T allele of the Enh1 variant results in reduced expression of RET, compared with the C allele, because the T allele disrupts binding to the transcription factor SOX10. We studied whether the A allele of Enh2 (Enh2-A) also affects RET gene expression.nnnMETHODSnWe evaluated the function of Enh1 and Enh2 using luciferase reporter assays with constructs that contained each allele, separately or in combination. We performed in silico analysis to identify transcription activators or repressors that bind to Enh2-C.nnnRESULTSnThe Enh1-T and the Enh2-A alleles reduced expression of the luciferase reporter gene. In silico analysis identified the sequence of Enh2-C and its surrounding sequence (ACGTG) as a potential binding site for the NXF-ARNT2 and SIM2-ARNT2 transcription factor heterodimers. The affinity of NXF-ARNT2 for Enh2-C was confirmed by electrophoresis mobility shift and supershift assays. Transfection of neuroblastoma cell lines with NXF-ARNT2 or SIM2-ARNT2 increased and decreased expression of RET, respectively.nnnCONCLUSIONSnMore than one SNP on an associated haplotype can influence gene expression and ultimately disease phenotype. Binding of the transcription factors NXF, ARNT2, and SIM2 to RET depend on the RET polymorphism of Enh2 and affect RET expression and the development of Hirschsprungs disease.

The intrinsic innervation of the lung is derived from neural crest cells as shown by optical projection tomography in Wnt1-Cre;YFP reporter mice

Within the embryonic lung, intrinsic nerve ganglia, which innervate airway smooth muscle, are required for normal lung development and function. We studied the development of neural crest‐derived intrinsic neurons within the embryonic mouse lung by crossing Wnt1‐Cre mice with R26R‐EYFP reporter mice to generate double transgenic mice that express yellow fluorescent protein (YFP) in all neural crest cells (NCCs) and their derivatives. In addition to utilizing conventional immunohistochemistry on frozen lung sections, the complex organization of lung innervation was visualized in three dimensions by combining the genetic labelling of NCCs with optical projection tomography, a novel imaging technique that is particularly useful for the 3D examination of developing organs within embryos. YFP‐positive NCCs migrated into the mouse lung from the oesophagus region at embryonic dayu200310.5. These cells subsequently accumulated around the bronchi and epithelial tubules of the lung and, as shown by 3D lung reconstructions with optical projection tomography imaging, formed an extensive, branching network in association with the developing airways. YFP‐positive cells also colonized lung maintained in organotypic culture, and responded in a chemoattractive manner to the proto‐oncogene, rearranged during transfection (RET) ligand, glial‐cell‐line‐derived neurotrophic factor (GDNF), suggesting that the RET signalling pathway is involved in neuronal development within the lung. However, when the lungs of Ret−/− and Gfrα1−/− embryos, deficient in the RET receptor and GDNF family receptor α 1 (GFRα1) co‐receptor respectively, were examined, no major differences in the extent of lung innervation were observed. Our findings demonstrate that intrinsic neurons of the mouse lung are derived from NCCs and that, although implicated in the development of these cells, the role of the RET signalling pathway requires further investigation.

Genetics of human enteric neuropathies

Knowledge of molecular mechanisms that underlie development of the enteric nervous system has greatly expanded in recent decades. Enteric neuropathies related to aberrant genetic development are thus becoming increasingly recognized. There has been no recent review of these often highly morbid disorders. This review highlights advances in knowledge of the molecular pathogenesis of these disorders from a clinical perspective. It includes diseases characterized by an infantile aganglionic Hirschsprung phenotype and those in which structural abnormalities are less pronounced. The implications for diagnosis, screening and possible reparative approaches are presented.

Cow's Milk Challenge Increases Weakly Acidic Reflux in Children with Cow's Milk Allergy and Gastroesophageal Reflux Disease

OBJECTIVEnTo assess and compare the pattern of reflux in a selected population of infants with cows milk (CM) allergy (CMA) and suspected gastroesophageal reflux disease (GERD) while on dietary exclusion and following challenge with CM.nnnSTUDY DESIGNnSeventeen children (median age: 14 months) with a proven diagnosis of CMA and suspected GERD underwent 48-hour multichannel intraluminal impedance-pH monitoring. For the first 24 hours, the infants were kept on amino acid-based formula, and for the subsequent 24 hours, they were challenged with CM.nnnRESULTSnThe total reflux episodes and the number of weakly acidic episodes were higher during CM challenge compared with the amino acid-based formula period [total reflux episodes: 105 (58-127.5) vs 65 (39-87.5), Pxa0<xa0.001; weakly acidic episodes: 53 (38.5-60.5) vs 19 (13-26.5), P < .001; median (25th-75th)]. No differences were found for either acid or weakly alkaline episodes (not significant). The number of weakly acidic episodes reaching the proximal, mid, and distal esophagus was higher during CM challenge (P < .001). No differences were found in either acid exposure time or number of long-lasting episodes (not significant).nnnCONCLUSIONSnIn children with CMA and suspected GERD, CM exposure increases the number of weakly acidic reflux episodes. CM challenge during 48-hour multichannel intraluminal impedance-pH monitoring identifies a subgroup of patients with allergen-induced reflux, and in selected cases of children with CMA in whom GERD is suspected, its use could be considered as part of diagnostic work-up.

Microporous collagen spheres produced via thermally induced phase separation for tissue regeneration

Collagen is an abundant protein found in the extracellular matrix of many tissues. Due to its biocompatibility, it is a potentially ideal biomaterial for many tissue engineering applications. However, harvested collagen often requires restructuring into a three-dimensional matrix to facilitate applications such as implantation into poorly accessible tissue cavities. The aim of the current study was to produce a conformable collagen-based scaffold material capable of supporting tissue regeneration for use in wound repair applications. Microporous collagen spheres were prepared using a thermally induced phase separation (TIPS) technique and their biocompatibility was assessed. The collagen spheres were successfully cross-linked with glutaraldehyde vapour, rendering them mechanically more stable. When cultured with myofibroblasts the collagen spheres stimulated a prolonged significant increase in secretion of the angiogenic growth factor, vascular endothelial growth factor (VEGF), compared with cells alone. Control polycaprolactone (PCL) spheres failed to stimulate a similar prolonged increase in VEGF secretion. An enhanced angiogenic effect was also seen in vivo using the chick embryo chorioallantoic membrane assay, where a significant increase in the number of blood vessels converging towards collagen spheres was observed compared with control PCL spheres. The results from this study indicate that microporous collagen spheres produced using TIPS are biologically active and could offer a novel conformable scaffold for tissue regeneration in poorly accessible wounds.

Gastrointestinal endoscopy and mucosal biopsy in the first year of life: indications and outcome.

Objectives: Lower threshold and widening indications for paediatric gastrointestinal endoscopy have resulted in a significant increase in the numbers of endoscopic procedures performed in infants. Despite this, knowledge of gastrointestinal mucosal findings in this age group is limited and data on the clinical usefulness of endoscopy are lacking. Methods: All of the children younger than 1 year referred to a single tertiary paediatric gastroenterology unit during the period June 1987 to August 2007 who underwent gastrointestinal endoscopy were identified and the clinical indications and histological outcomes were reviewed. Results: A total of 933 gastroesophageal duodenoscopies and 439 colonoscopies were performed in 1024 cases in a total of 823 infants. In order of frequency, clinical indications were diarrhoea (51%), failure to thrive (41.2%), symptoms of reflux (27.1%), and rectal bleeding (8.5%). Mucosal biopsies were insufficient for assessment in only 2.4% of cases. Mucosal histology was normal in 33.8%, whereas histological abnormalities were identified in 63.8%. Specific histological diagnoses included microvillous inclusion disease, autoimmune enteropathy, graft-versus-host disease post–bone marrow transplantation, tufting enteropathy, and disaccharidase deficiency. There was only 1 colonic perforation complicating endoscopy in a total of 889 cases for which relevant information was available (0.1%). Conclusions: In two-thirds of cases, histological abnormalities were detected that influenced management following endoscopic examination and mucosal biopsy in infants. Endoscopy with biopsies is a greatly informative test with low failure and complication rates in the first year of life.