Christa N. Grant

Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function

Short bowel syndrome (SBS) is a devastating condition in which insufficient small intestinal surface area results in malnutrition and dependence on intravenous parenteral nutrition. There is an increasing incidence of SBS, particularly in premature babies and newborns with congenital intestinal anomalies. Tissue-engineered small intestine (TESI) offers a therapeutic alternative to the current standard treatment, intestinal transplantation, and has the potential to solve its biggest challenges, namely donor shortage and life-long immunosuppression. We have previously demonstrated that TESI can be generated from mouse and human small intestine and histologically replicates key components of native intestine. We hypothesized that TESI also recapitulates native small intestine function. Organoid units were generated from mouse or human donor intestine and implanted into genetically identical or immunodeficient host mice. After 4 wk, TESI was harvested and either fixed and paraffin embedded or immediately subjected to assays to illustrate function. We demonstrated that both mouse and human tissue-engineered small intestine grew into an appropriately polarized sphere of intact epithelium facing a lumen, contiguous with supporting mesenchyme, muscle, and stem/progenitor cells. The epithelium demonstrated major ultrastructural components, including tight junctions and microvilli, transporters, and functional brush-border and digestive enzymes. This study demonstrates that tissue-engineered small intestine possesses a well-differentiated epithelium with intact ion transporters/channels, functional brush-border enzymes, and similar ultrastructural components to native tissue, including progenitor cells, whether derived from mouse or human cells.

Adult zebrafish intestine resection: a novel model of short bowel syndrome, adaptation, and intestinal stem cell regeneration

Loss of significant intestinal length from congenital anomaly or disease may lead to short bowel syndrome (SBS); intestinal failure may be partially offset by a gain in epithelial surface area, termed adaptation. Current in vivo models of SBS are costly and technically challenging. Operative times and survival rates have slowed extension to transgenic models. We created a new reproducible in vivo model of SBS in zebrafish, a tractable vertebrate model, to facilitate investigation of the mechanisms of intestinal adaptation. Proximal intestinal diversion at segment 1 (S1, equivalent to jejunum) was performed in adult male zebrafish. SBS fish emptied distal intestinal contents via stoma as in the human disease. After 2 wk, S1 was dilated compared with controls and villus ridges had increased complexity, contributing to greater villus epithelial perimeter. The number of intervillus pockets, the intestinal stem cell zone of the zebrafish increased and contained a higher number of bromodeoxyuridine (BrdU)-labeled cells after 2 wk of SBS. Egf receptor and a subset of its ligands, also drivers of adaptation, were upregulated in SBS fish. Igf has been reported as a driver of intestinal adaptation in other animal models, and SBS fish exposed to a pharmacological inhibitor of the Igf receptor failed to demonstrate signs of intestinal adaptation, such as increased inner epithelial perimeter and BrdU incorporation. We describe a technically feasible model of human SBS in the zebrafish, a faster and less expensive tool to investigate intestinal stem cell plasticity as well as the mechanisms that drive intestinal adaptation.

Tissue engineering: a promising therapeutic approach to necrotizing enterocolitis.

Tissue engineering is a promising potential candidate for treating intestinal failure resulting from necrotizing enterocolitis. This requires the acquisition, preparation and implantation of autologous organoid units. This may be affected by the complexities of periods of storage of viable donor tissue and delayed implantation. This chapter addresses the development, methodology, and application of tissue-engineered intestine in the experimental and clinical setting. Tissue engineering has the potential of avoiding the inherent toxicities of intestinal transplantation and prolonged immunosuppression.

Prolonged Absence of Mechanoluminal Stimulation in Human Intestine Alters the Transcriptome and Intestinal Stem Cell Niche

Background & Aims For patients with short-bowel syndrome, intestinal adaptation is required to achieve enteral independence. Although adaptation has been studied extensively in animal models, little is known about this process in human intestine. We hypothesized that analysis of matched specimens with and without luminal flow could identify new potential therapeutic pathways. Methods Fifteen paired human ileum samples were collected from children aged 2–20 months during ileostomy-reversal surgery after short-segment intestinal resection and diversion. The segment exposed to enteral feeding was denoted as fed, and the diverted segment was labeled as unfed. Morphometrics and cell differentiation were compared histologically. RNA Sequencing and Gene Ontology Enrichment Analysis identified over-represented and under-represented pathways. Immunofluorescence staining and Western blot evaluated proteins of interest. Paired data were compared with 1-tailed Wilcoxon rank-sum tests with a P value less than .05 considered significant. Results Unfed ileum contained shorter villi, shallower crypts, and fewer Paneth cells. Genes up-regulated by the absence of mechanoluminal stimulation were involved in digestion, metabolism, and transport. Messenger RNA expression of LGR5 was significantly higher in unfed intestine, accompanied by increased levels of phosphorylated signal transducer and activator of transcription 3 protein, and CCND1 and C-MYC messenger RNA. However, decreased proliferation and fewer LGR5+, OLFM4+, and SOX9+ intestinal stem cells (ISCs) were observed in unfed ileum. Conclusions Even with sufficient systemic caloric intake, human ileum responds to the chronic absence of mechanoluminal stimulation by up-regulating brush-border enzymes, transporters, structural genes, and ISC genes LGR5 and ASCL2. These data suggest that unfed intestine is primed to replenish the ISC population upon re-introduction of enteral feeding. Therefore, the elucidation of pathways involved in these processes may provide therapeutic targets for patients with intestinal failure. RNA sequencing data are available at Gene Expression Omnibus series GSE82147.

Doxycycline Sclerotherapy Is Superior in the Treatment of Pediatric Lymphatic Malformations

PURPOSE To evaluate efficacy of sclerotherapy with doxycycline versus sodium tetradecyl sulfate (STS) for treatment of macrocystic and mixed lymphatic malformations (LMs). MATERIALS AND METHODS This single-center retrospective review identified 41 children (17 boys; 24 girls; age range, 1 month to 15.4 y) who underwent sclerotherapy with doxycycline (n = 32) or STS (n = 9) for macrocystic (n = 31) or mixed (n = 10) LMs. There were 114 treatments performed, averaging 2.8 treatments (range, 1-8 treatments) per patient. Average follow-up time was 10 months (range, 1-59 months). Clinical response was deemed excellent or moderate if > 90% or > 50% of LMs resolved based on visual estimate. RESULTS With doxycycline, 87% of patients (28 of 32) had excellent or moderate response with an average of 2.8 treatments (range, 1-7 treatments); 13% required subsequent resection. With 3% STS monotherapy, only 55% of patients (5 of 9) had excellent or moderate response with an average of 2.8 treatments (range, 1-8 treatments), and 33% required subsequent resection. Significantly fewer patients treated with STS responded well compared with patients treated with doxycycline (P = .03). Patients treated with STS had significantly longer follow-up than patients treated with doxycycline (27 months vs 6 months, P = .0001). CONCLUSIONS Doxycycline monotherapy resulted in a high rate of excellent clinical outcomes after a few treatments without increased need for subsequent operative resection. These results support use of doxycycline sclerotherapy as primary treatment for macrocystic and mixed LMs in children.

A case of congenital pyloric atresia with dystrophic epidermolysis bullosa

Pyloric atresia with epidermolysis bullosa (EB) dystrophica is a rare entity that may not be immediately recognized. We describe the fourth confirmed case of pyloric atresia associated with the dystrophic subtype of EB diagnosed by standard pathologic measures, and discuss the clinical disease features and recent advances in the pathophysiology.

Routine contrast enema is not required for all infants prior to ostomy reversal: A 10-year single-center experience

INTRODUCTION The incidence of intestinal stricture is low for most conditions requiring a primary small bowel stoma in infants. Routine performance of contrast enemas (CE) prior to stoma closure adds cost and radiation exposure. We hypothesized that routine CE prior to ostomy reversal is not necessary in all infants, and sought to identify a subset of patients who may benefit from preoperative CE. METHODS Medical records of infants under age 1 (N=161) undergoing small bowel stoma reversal at a single institution between 2003 and 2013 were retrospectively reviewed. Students T-test was used to compare groups. RESULTS Contrast enemas were performed on 80% of all infants undergoing small bowel ostomy reversal during the study period. Infants with necrotizing enterocolitis (NEC) were more likely to have a CE than those with intestinal atresia (p=0.03) or those with all other diagnoses combined (p=0.03). Nine strictures were identified on CE. Of those, 8 (89%) were in patients with NEC, and only 4 were clinically significant and required operative resection. The overall relevant stricture rate was 2.5%. No patient that underwent ostomy takedown without CE had a stricture diagnosed intraoperatively or an unrecognized stricture that presented clinically after stoma takedown. CONCLUSIONS Routine CE is not required prior to small bowel ostomy reversal in infants. We recommend judicious use of enema studies in patients with NEC and high likelihood of stricture.

Intestinal Regeneration: The Stem Cell Approach

The ultimate goal of therapies for intestinal failure is to replace the structure and function of the absent region of the intestine. Intestinal failure is the inability to independently maintain nutrition via enteral absorption and has many causes, including genetic and metabolic abnormalities. Short bowel syndrome (SBS) is one form of intestinal failure caused by the anatomical loss of intestinal length. While patients with SBS are able to survive with intravenous nutrition, quality of life is poor, and morbidity is high. Liver failure and sepsis are the most common morbidities. Intestine and multivisceral transplantation have salvaged many children but also come at a high human and financial cost. With complications from necessary immunosuppression and significant graft failure rates, alternative therapies are needed. Recent advances in intestinal stem cell (ISC) and developmental biology have led to the identification and analysis of multiple putative ISCs and progenitor cells. Concurrent growth in the fields of material bioengineering and cell biology has augmented the growth of the field of tissue engineering. By replicating and manipulating the normal developmental and regenerative processes, it has been possible to grow tissue-engineered intestine. Further study to refine the processes and generate larger quantities of engineered tissue will be necessary in order to employ tissue-engineered intestine as a viable future strategy for intestinal replacement in patients with intestinal failure.

Mo1843 Parenteral Fish Oil Reverses Cholestasis in Parenteral Nutrition Associated Liver Disease

from liver failure. 2) Investigate the role of microglial activation and recruitment in the pathogenesis of HE. Methods: Male C57Bl/6 mice were injected with 100 mg/kg of the hepatotoxin azoxymethane (AOM) to induce HE. Mice were monitored for signs of cognitive impairment and brains were collected and dissected prior to neurological symptoms, at the onset of minor and major ataxia, and at coma. In parallel, mice were then injected with 100mg/kg/day of CCR2 antagonist or CCR4 antagonist for three days prior to the injection of AOM. Tissue was collected at the time that coma was reached. Immunofluorescence, immunoblotting, and real time PCR was performed for CCL2, CCR2, and CCR4. Microglia activation was assessed by immunofluorescence against the microglia marker Iba1. Results: CCL2 mRNA expression is greatly increased prior to the onset of neurological decline in the cortex and remains elevated when compared to vehicle-treated controls. CCL2 protein was significantly elevated in the cortex but not the cerebellum as determined by immunoblotting. Immunofluorescence validated this effect and found localized immunoreactivity of CCL2 with the neuron marker NeuN. Correlated with elevated CCL2 expression was increased microglia activation as demonstrated by Iba1 immunoreactivity. Treatment with CCR2 and CCR4 antagonists, which inhibit CCL2 activity, reducedmicroglia activation and neurological decline of AOM mice. Conclusions: The data demonstrates that CCL2 is upregulated during HE and inhibiting its activity via CCR2 or CCR4 antagonists slows HE progression. This supports that during HE neurons may release CCL2 to recruit and activate microglia leading to pathological inflammation.