Allan M. Goldstein

The Mechanism of Excessive Intestinal Inflammation in Necrotizing Enterocolitis: An Immature Innate Immune Response

Necrotizing enterocolitis (NEC) is a devastating neonatal intestinal inflammatory disease, occurring primarily in premature infants, causing significant morbidity and mortality. The pathogenesis of NEC is associated with an excessive inflammatory IL-8 response. In this study, we hypothesized that this excessive inflammatory response is related to an immature expression of innate immune response genes. To address this hypothesis, intestinal RNA expression analysis of innate immune response genes was performed after laser capture microdissection of resected ileal epithelium from fetuses, NEC patients and children and confirmed in ex vivo human intestinal xenografts. Changes in mRNA levels of toll-like receptors (TLR)-2 and -4, their signaling molecules and transcription factors (MyD88, TRAF-6 and NFκB1) and negative regulators (SIGIRR, IRAK-M, A-20 and TOLLIP) and the effector IL-8 were characterized by qRT-PCR. The expression of TLR2, TLR4, MyD88, TRAF-6, NFκB1 and IL-8 mRNA was increased while SIGIRR, IRAK-M, A-20 and TOLLIP mRNA were decreased in fetal vs. mature human enterocytes and further altered in NEC enterocytes. Similar changes in mRNA expression were observed in immature, but not mature, human intestinal xenografts. Confirmation of gene expression was also validated with selective protein measurements and with suggested evidence that immature TRL4 enterocyte surface expression was internalized in mature enterocytes. Cortisone, an intestinal maturation factor, treatment corrected the mRNA differences only in the immature intestinal xenograft. Using specific siRNA to attenuate expression of primary fetal enterocyte cultures, both TOLLIP and A-20 were confirmed to be important when knocked down by exhibiting the same excessive inflammatory response seen in the NEC intestine. We conclude that the excessive inflammatory response of the immature intestine, a hallmark of NEC, is due to a developmental immaturity in innate immune response genes.

BMP signaling is necessary for neural crest cell migration and ganglion formation in the enteric nervous system

The enteric nervous system (ENS) is derived from neural crest cells that migrate along the gastrointestinal tract to form a network of neurons and glia that are essential for regulating intestinal motility. Despite the number of genes known to play essential roles in ENS development, the molecular etiology of congenital disorders affecting this process remains largely unknown. To determine the role of bone morphogenetic protein (BMP) signaling in ENS development, we first examined the expression of bmp2, bmp4, and bmprII during hindgut development and find these strongly expressed in the ENS. Moreover, functional BMP signaling, demonstrated by the expression of phosphorylated Smad1/5/8, is present in the enteric ganglia. Inhibition of BMP activity by noggin misexpression within the developing gut, both in ovo and in vitro, inhibits normal migration of enteric neural crest cells. BMP inhibition also leads to hypoganglionosis and failure of enteric ganglion formation, with crest cells unable to cluster into aggregates. Abnormalities of migration and ganglion formation are the hallmarks of two human intestinal disorders, Hirschsprungs disease and intestinal neuronal dysplasia. Our results support an essential role for BMP signaling in these aspects of ENS development and provide a basis for further investigation of these proteins in the etiology of neuro-intestinal disorders.

Building a brain in the gut: development of the enteric nervous system

The enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract, is an essential component of the gut neuromusculature and controls many aspects of gut function, including coordinated muscular peristalsis. The ENS is entirely derived from neural crest cells (NCC) which undergo a number of key processes, including extensive migration into and along the gut, proliferation, and differentiation into enteric neurons and glia, during embryogenesis and fetal life. These mechanisms are under the molecular control of numerous signaling pathways, transcription factors, neurotrophic factors and extracellular matrix components. Failure in these processes and consequent abnormal ENS development can result in so‐called enteric neuropathies, arguably the best characterized of which is the congenital disorder Hirschsprung disease (HSCR), or aganglionic megacolon. This review focuses on the molecular and genetic factors regulating ENS development from NCC, the clinical genetics of HSCR and its associated syndromes, and recent advances aimed at improving our understanding and treatment of enteric neuropathies.

Extracardiac Anomalies in the Heterotaxy Syndromes With Focus on Anomalies of Midline-Associated Structures

The extracardiac defects in patients with heterotaxy have not been examined as extensively as cardiac defects. We found a high incidence of midline-associated defects in 160 autopsied cases of heterotaxy (asplenia, polysplenia, or single right-sided spleen). Fifty-two percent of patients with left-sided polysplenia had a midline-associated defect, as did 45% of those with asplenia. Most common were musculoskeletal or genitourinary anomalies, as well as cleft palate. Fused adrenal glands and anal stenosis or atresia occurred exclusively among patients with asplenia. A midline anomaly was twice as likely to be detected on complete autopsy than from clinical findings alone. Linkage studies should take into account that affected subjects may have isolated subclinical midline defects. The high incidence of midline-associated defects supports the theory that the midline plays a critical role in establishing left-right asymmetry in the body. Comparison of these defects with mouse models of laterality defects suggests that mutations that disrupt the transforming growth factor beta pathway may result in heterotaxy.

Intestinal malrotation: Varied clinical presentation from infancy through adulthood

BACKGROUND The purpose of this study was to determine the incidence and clinical presentation of intestinal malrotation from infancy through adulthood by examining the experience of a single institution caring for patients of all ages with this condition. METHODS We conducted a retrospective review on all patients diagnosed with intestinal malrotation at Massachusetts General Hospital between 1992 and 2009. Patient demographics, clinical history, diagnostic tests, operative procedures, and outcome variables were recorded. Patients were divided into 3 age groups: infants (<1 year), children (1-18 years), and adults (18 years). RESULTS We identified 170 patients, of whom 31% were infants, 21% were children, and 48% were adults. Infants nearly always presented with emesis (93%), whereas adults most commonly presented with abdominal pain (87%), and less often with emesis (37%) or nausea (31%). The incidence of volvulus declined with age, from 37% to 22% to 12%, in each of the 3 age groups, respectively. Although infants were most often diagnosed within hours or days of symptom onset, 59% of children and 32% of adults experienced symptoms for years before diagnosis. Upper gastrointestinal series was the most common imaging study performed in infants and children, but was replaced by abdominal computed tomography in adults. All infants and children underwent a Ladds procedure, compared with only 61% of adults. The majority of patients experienced resolution of symptoms after operative intervention, although this decreased slightly with age. CONCLUSION Intestinal malrotation can occur in patients of any age and, in contrast with traditional teaching, nearly half of these patients may present during adulthood. An increased awareness of this entity and an understanding of its varied presentation at different ages may reduce time to diagnosis and improve patient outcome.

Bone morphogenetic protein signaling pathway plays multiple roles during gastrointestinal tract development

The bone morphogenetic protein (BMP) signaling pathway plays an essential role during gastrointestinal (GI) tract development in vertebrates. In the present study, we use an antibody that recognizes the phosphorylated and activated form of Smad1, 5, and 8 to examine (by immunohistochemistry) the endogenous patterns of BMP signaling pathway activation in the developing GI tract. We show that the endogenous BMP signaling pathway is activated in the mesoderm, the endoderm, and the enteric nervous system (ENS) of the developing chick GI tract and is more widespread than BMP ligand expression patterns. Using an avian‐specific retroviral misexpression technique to activate or inhibit BMP signaling pathway activity in the mesoderm of the gut, we show that BMP activity is required for the pattern, the development, and the differentiation of all three tissue types of the gut: mesoderm (that forms the visceral smooth muscle), endoderm (that forms the epithelium), and ectoderm (that forms the ENS). These results demonstrate that BMP signaling is activated in all the tissue layers of the GI tract during the development and plays a role during interactions and reciprocal communications of these tissue layers. Developmental Dynamics 234:312–322, 2005.

Extracorporeal Membrane Oxygenation for Nonneonatal Acute Respiratory Failure: The Massachusetts General Hospital Experience From 1990 to 2008

OBJECTIVE To determine the efficacy of extracorporeal membrane oxygenation (ECMO) for nonneonatal acute respiratory failure. DESIGN Single-institution, retrospective medical record review from February 1990 to March 2008. SETTING Tertiary care hospital. PATIENTS Eighty-one nonneonatal patients (mean age, 23 years; age range, 2 months to 61 years) with acute respiratory failure who had failed maximal ventilator support received ECMO therapy between 1990 and 2008. Patients were grouped into 6 categories based on diagnosis: sepsis (n = 8), bacterial or fungal pneumonia (n = 15), viral pneumonia (n = 9), trauma or burn (n = 10), immunocompromise (n = 15), and other (n = 24). Main Outcome Measure Survival to hospital discharge. RESULTS Overall survival was 53%. Survival was highest in patients with viral pneumonia (78%), followed by bacterial pneumonia (53%), sepsis syndrome (44%), and immunocompromise (40%). Patients treated following trauma or burns had the lowest survival (33%). The average age was 19 years for survivors as compared with 27 years for nonsurvivors. Survival was lower in patients with multiple organ failure as compared with those with single organ failure (33% vs 60%, respectively), in patients who experienced mechanical ventilation for longer than 10 days prior to the initiation of ECMO as compared with those who received ventilatory support for less than 10 days prior to the initiation of ECMO (31% vs 57%, respectively), and in patients requiring more than 400 hours of ECMO support as compared with those requiring less than 400 hours of ECMO support (42% vs 55%, respectively). CONCLUSIONS Therapy with ECMO may provide a survival benefit in carefully selected patients with nonneonatal acute respiratory failure who have failed maximal ventilator support. Nonneonatal survival with ECMO therapy is strongly dependent on diagnosis, with the highest survival seen in those with viral or bacterial pneumonia. Older age, multiple organ failure, prolonged ventilation prior to ECMO initiation, and long ECMO runs are associated with decreased survival.

Endothelial cells promote migration and proliferation of enteric neural crest cells via β1 integrin signaling

Enteric neural crest-derived cells (ENCCs) migrate along the intestine to form a highly organized network of ganglia that comprises the enteric nervous system (ENS). The signals driving the migration and patterning of these cells are largely unknown. Examining the spatiotemporal development of the intestinal neurovasculature in avian embryos, we find endothelial cells (ECs) present in the gut prior to the arrival of migrating ENCCs. These ECs are patterned in concentric rings that are predictive of the positioning of later arriving crest-derived cells, leading us to hypothesize that blood vessels may serve as a substrate to guide ENCC migration. Immunohistochemistry at multiple stages during ENS development reveals that ENCCs are positioned adjacent to vessels as they colonize the gut. A similar close anatomic relationship between vessels and enteric neurons was observed in zebrafish larvae. When EC development is inhibited in cultured avian intestine, ENCC migration is arrested and distal aganglionosis results, suggesting that ENCCs require the presence of vessels to colonize the gut. Neural tube and avian midgut were explanted onto a variety of substrates, including components of the extracellular matrix and various cell types, such as fibroblasts, smooth muscle cells, and endothelial cells. We find that crest-derived cells from both the neural tube and the midgut migrate avidly onto cultured endothelial cells. This EC-induced migration is inhibited by the presence of CSAT antibody, which blocks binding to beta1 integrins expressed on the surface of crest-derived cells. These results demonstrate that ECs provide a substrate for the migration of ENCCs via an interaction between beta1 integrins on the ENCC surface and extracellular matrix proteins expressed by the intestinal vasculature. These interactions may play an important role in guiding migration and patterning in the developing ENS.

Femoral venous access is safe in burned children: An analysis of 224 catheters☆☆☆★★★

OBJECTIVE To document the incidence of septic and mechanical complications associated with femoral venous catheters in a subgroup of patients thought to be at particularly high risk of both: young children with large burns. DESIGN An analysis of data collected prospectively on all femoral venous catheters placed during a 4-year period at a regional pediatric burn facility. RESULTS There were 224 femoral catheters placed in 86 children with an average age of 5.3 +/- 5.1 years and an average burn size of 38% +/- 23%. Catheters were left in place for a mean duration of 5.7 days. Catheter-related sepsis occurred with 4.9% of the catheters, and mechanical complications occurred in 3.5% of the patients. There was no statistically significant association between the risk of catheter sepsis and the placement of catheters through burned versus unburned skin. Similarly, the risk of sepsis was equivalent between lines placed over a guide wire and those placed of a new site. CONCLUSION Femoral venous catheters are safe in burned children and are associated with a low incidence of infectious and mechanical complications.

Analysis of gastrointestinal physiology using a novel intestinal transit assay in zebrafish

Abstract  Gastrointestinal function depends upon coordinated contractions to mix and propel food through the gut. Deregulation of these contractions leads to alterations in the speed of material transit through the gut, with potentially significant consequences. We have developed a method for visualizing intestinal transit, the physiological result of peristaltic contractions, in larval zebrafish. This method allows direct, non‐invasive observation of luminal content as it traverses the gut. Using this method, we characterized gastrointestinal transit in zebrafish larvae at 7 days postfertilization. In addition, we used this transit assay to assess the physiological consequences of reduced or absent enteric neurones on intestinal transit in larval zebrafish. This may facilitate the use of the zebrafish for investigating the effect of compounds and candidate genes on gastrointestinal motility.