Development of potentially life-threatening enterocolitis is the most frequent complication in children with Hirschsprung disease (HSCR), even after definitive corrective surgery. Intestinal microbiota likely contribute to the etiology of enterocolitis, so the aim of this study was to compare the fecal bacterial and fungal communities of children who developed Hirschsprung-associated enterocolitis (HAEC) with HSCR patients who had never had enterocolitis. Eighteen Hirschsprung patients who had completed definitive surgery were enrolled: 9 had a history of HAEC and 9 did not. Fecal DNA was isolated and 16S and ITS-1 regions sequenced using Next Generation Sequencing and data analysis for species identification. The HAEC group bacterial composition showed a modest reduction in Firmicutes and Verrucomicrobia with increased Bacteroidetes and Proteobacteria compared with the HSCR group. In contrast, the fecal fungi composition of the HAEC group showed marked reduction in diversity with increased Candida sp., and reduced Malassezia and Saccharomyces sp. compared with the HSCR group. The most striking finding within the HAEC group is that the Candida genus segregated into “high burden” patients with 97.8% C. albicans and 2.2% C. tropicalis compared with “low burden” patients 26.8% C. albicans and 73% C. tropicalis. Interestingly even the low burden HAEC group had altered Candida community structure with just two species compared to more diverse Candida populations in the HSCR patients. This is the first study to identify Candida sp. as potentially playing a role in HAEC either as expanded commensal species as a consequence of enterocolitis (or treatment), or possibly as pathobioants contributing to the pathogenesis of HAEC. These findings suggest a dysbiosis in the gut microbial ecosystem of HAEC patients, such that there may be dominance of fungi and bacteria predisposing patients to development of HAEC.

Ty elements of Saccharomyces cerevisiae are long terminal repeat (LTR) retroelements related to retroviruses. Normal levels of Ty1 transposition require Dbr1p, a cellular enzyme that cleaves 2'-5' RNA bonds. We show that Ty1 RNAs lacking identifiable 5' ends accumulate in virus-like particles (VLPs) in dbr1 mutants. Debranching this RNA in vitro with Dbr1p creates an uncapped version of the normal Ty1 RNA 5' end. We show that the 5' nucleotide (nt) of Ty1 RNA forms a 2'-5' bond with a nt near the 3' end of the same RNA, creating a lariat. The properties of the lariat suggest it forms by a novel mechanism and that branching and debranching may play roles in Ty1 reverse transcription at the minus-strand transfer step.

Purpose The aim of the study was to characterize enterocolitis in the Ednrb-null (Ednrb−/−) mouse with aganglionosis of the colon and to develop and validate a semiquantitative histopathologic grading system to assess enterocolitis. Methods We isolated colon and ileal specimens of Ednrb−/− and control mice (Ednrb+/+) and performed histochemical staining (H&E) on tissue sections. After establishing inflammation grading criteria, 2 blinded pathologists independently assessed the severity and depth of inflammation of proximal colon segments on 2 separate occasions. Interclass correlations (ICCs) and coefficient of variation (CV) were calculated to determine interrater and intrarater agreement. We then prospectively applied the enterocolitis grading system to Ednrb−/− mice that became clinically ill. A cohort of Ednrb−/− mice were observed until they developed clinical illness, at which time they were euthanized and had multiple organ homogenates cultured for bacteria, and colon and small bowel were histopathologically graded for enterocolitis. Spearman’s rank correlations comparing enterocolitis scores with level of bacteremia were performed. Results Intra- and interrater ICCs of the histologic scoring system were satisfactory (0.61 and 0.94, respectively), as were intra- and interrater CVs (18% and 9%, respectively). Of the Ednrb−/− mice, 65% developed bacteremia. Those with bacteremia had significantly higher enterocolitis scores than those without bacteremia (P < .01). Ednrb−/− mice that developed bacteremia showed a strong positive correlation between total enterocolitis scores and number of bacterial colony forming units in peritoneal lavage, liver, kidney, and aerobic spleen. Conclusions The Ednrb−/− mouse with aganglionosis develops enterocolitis and has features similar to Hirschsprung-associated enterocolitis in humans. Our grading system is a reliable way to assess enterocolitis. By performing microsurgical pull-through, we can now perform controlled, hypothesis-driven, mechanistic studies to evaluate etiologic factors affecting enterocolitis in the Ednrb−/− mouse.

Purpose Children with Hirschsprung disease (HD) who have a history of enterocolitis (HAEC) have a shift in colonic microbiota, many of which are necessary for short chain fatty acid (SCFA) production. As SCFAs play a critical role in colonic mucosal preservation, we hypothesized that fecal SCFA composition is altered in children with HAEC. Methods A multicenter study enrolled 18 HD children, abstracting for history of feeding, antibiotic/probiotic use, and enterocolitis symptoms. HAEC status was determined per Pastor et al. criteria (12). Fresh feces were collected for microbial community analysis via 16S sequencing as well as SCFA analysis by gas chromatography–mass spectrometry. Results Nine patients had a history of HAEC, and nine had never had HAEC. Fecal samples from HAEC children showed a 4-fold decline in total SCFA concentration vs. non-HAEC HD patients. We then compared the relative composition of individual SCFAs and found reduced acetate and increased butyrate in HAEC children. Finally, we measured relative abundance of SCFA-producing fecal microbiota. Interestingly, 10 of 12 butyrate-producing genera as well as 3 of 4 acetate-producing genera demonstrated multi-fold expansion. Conclusion Children with HAEC history have reduced fecal SCFAs and altered SCFA profile. These findings suggest a complex interplay between the colonic metabolome and changes in microbiota, which may influence the pathogenesis of HAEC.

Potentially life-threatening enterocolitis is the most frequent complication in children with colonic aganglionosis (Hirschsprung disease, HSCR), and little is known about the mechanisms leading to enterocolitis. Splenic lymphopenia has been reported in the Endothelin Receptor B (Ednrb)-null mouse model of HSCR that develops enterocolitis. In this study, we sought to identify molecular mechanisms underlying this immune phenotype. We employed the Ednrb−/− mouse, and the knockout of its ligand, Edn3 (Edn3−/−). The major finding is that enterocolitis in the Ednrb−/− and Edn3−/− mice lead to thymic involution, splenic lymphopenia, and suppression of B lymphopoiesis as a consequence of colonic aganglionosis, not an intrinsic Edn3-Ednrb signaling defect directly affecting the lymphoid organs. We showed that adoptive transfer of Ednrb−/− marrow repopulated the RAG2-null mice marrow, thymus and spleen without development of enterocolitis. We identified the glucocorticoid corticosterone, as a potential mediator of the immune phenotype. This previously unrecognized pattern of immune abnormalities in mouse is nearly identical to lymphoid depletion in neonatal sepsis during severe physiological stress, suggesting that the mouse model used here could be also used for sepsis studies.

Background Hirschsprung disease (HD) results from aganglionosis of the colon, is linked to acute and chronic enterocolitis (known as Hirschsprung-associated enterocolitis) despite successful corrective surgery, and can lead to bacteremia and even death. The genetic and molecular mechanisms underlying these disorders are largely unknown. Methods We developed a microsurgical corrective pull-through procedure in mice, and applied that to Ednrb−/− mice, which manifest aganglionic megacolon that is very similar to HD. Wild-type littermates (Ednrb+/+) also underwent identical surgery. At prespecified time points postoperatively, mice were sacrificed, and histopathologic analyses of intestinal inflammation were performed. Mice of both genotypes were sacrificed after the postoperative recovery period to determine if corrective surgery itself caused inflammation. Stooling patterns were assessed as well to determine if intestinal function normalized after surgery. Results: There was no difference in histopathological enterocolitis scores after recovery from surgery. Stooling patterns in Ednrb−/− and Ednrb+/+ mice were similar postoperatively, suggesting normalization of intestinal function. However, with time, approximately 40% of Ednrb−/− mice developed clinical illness consistent with enterocolitis. No control (Ednrb+/+) mice developed clinical enterocolitis. Histopathological enterocolitis scores in the 40% of Ednrb−/− mice that developed clinical enterocolitis postoperatively were significantly worse than those of healthy postoperative Ednrb−/− mice. In contrast, none of the Ednrb+/+ control mice exhibited postoperative long-term inflammation. Conclusions Microsurgical pull-through operation in Ednrb−/− mice produces a mouse model that closely resembles key features of Hirschsprung-associated enterocolitis, enabling controlled study of genetic and molecular mechanisms in Ednrb−/− mice and other genotypes that produce similar phenotypes.

Purpose The aim of the study was to describe and characterize a novel small spleen phenotype with splenic lymphopenia in the Ednrb-null (Ednrb−/−) mouse with aganglionosis known to also develop enterocolitis. Methods We compared spleen weight as a percent of body weight from Ednrb+/+, Ednrb+/−, and Ednrb−/− mice to quantify our initial observation. Splenic microarchitecture of Ednrb+/+ and Ednrb−/− mice was assessed using both H and E staining and immunofluorescence staining for CD45R+ (B cells) and CD3+ (T cells) on tissue sections. To identify and quantify cell type, flow cytometry for CD19+ (mature B cells), CD4+ and CD8+ (T cells) was performed on the splenocytes of Ednrb+/+ and Ednrb−/− mice and compared with student’s t test. A separate cohort of Ednrb+/+ and Ednrb−/− mice was killed and splenocytes were analyzed by flow cytometry, and proximal colon was histopathologically graded for enterocolitis. Spearman’s rank correlations comparing total splenocyte and CD19+ cell counts with enterocolitis scores were performed. Results We found that the mean spleen weight expressed as a percent of body weight for Ednrb+/+ and Ednrb−/− mice was 0.72 and 0.25%, respectively (P < 0.001), at 25 days of age. In addition, the Ednrb−/− spleens also had markedly abnormal splenic microarchitecture with lymphopenia, and relative reduction of B cells compared to T cells. FACS of splenocytes revealed a 5 to 20-fold reduction in total cell number, CD19+, CD4+, and CD8+ of the Ednrb−/− mice compared to the Ednrb+/+ littermates (P < 0.01). We also found a strong inverse correlation of total spleen and CD19+ cell counts with histopathological enterocolitis scores (rs = −0.43, P = 0.02), showing that mice with reduced cell counts also had increased severity of enterocolitis. Conclusion The small spleen immunophenotype in the Ednrb−/− mouse suggests that Ednrb-dependent signaling may be required for normal spleen development. These results raise the possibility that primary immune abnormalities may contribute at least in part to some enterocolitis. At present, our data suggest intriguing new potential explanations for HAEC in Hirschsprung patients.

Background/Purpose The study aimed to develop a mouse model of post-pullthrough Hirschsprung’s disease that will allow investigation of mechanisms that cause postoperative complications. Methods We developed a novel microsurgical pullthrough operation on Balb/C mice and evaluated its effect on growth rate and stooling pattern. Histologic assessment of the pullthrough colon was performed. The pullthrough operation was then performed on Ednrb−/− mice that have aganglionic megacolon and Ednrb+/+ littermate controls, and the outcomes compared. Results The Balb/C pullthrough group had 97% survival at 1 week and 70% survival at 2 weeks. Body weight of the pullthrough animals declined 15% in the first week after surgery and subsequently normalized. The stooling pattern showed consistently softer stools in the pullthrough group, but no difference in frequency compared to controls. Histopathologic analyses 4 weeks postoperatively showed well-healed coloanal anastomoses. Two-week survival after pullthrough surgery in Ednrb−/− and Ednrb+/+ mice was 50.0%, and 69.2%, respectively (P = NS). Increased mortality in the Ednrb−/− mice was related to the technical challenge of performing microsurgery on smaller-sized mice with poor baseline health status. Conclusions Our microsurgical pullthrough operation in mice is feasible and allows systematic investigations into potential mechanisms mediating post-pullthrough complications and poor long-term results in mouse models of Hirschsprung’s disease.