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Hirschsprung-associated enterocolitis (HAEC) represents a severe complication of Hirschsprung disease, characterized by intestinal barrier dysfunction and life-threatening inflammation. The present study systematically reviews the updated molecular mechanisms underlying HAEC pathogenesis, with particular focus on the tight junction (TJ) proteins claudin, occludin and zonula occludens protein 1 (ZO-1) and their interactions with the actin cytoskeleton. The present review demonstrates that dysregulation of claudin family members, particularly upregulation of pore-forming claudin-2 and downregulation of barrier-forming claudin-4, disrupts intestinal homeostasis. Occludin undergoes cytokine-mediated endocytosis through myosin light chain kinase (MLCK)/NF-κB signaling, while ZO-1 dysfunction impairs mechanical coupling between TJs and actin filaments. Furthermore, the present review identifies that inflammatory mediators, such as IL-1β, TNF-α and IFN-γ, trigger actin cytoskeleton remodeling via the cofilin phosphorylation cycle and the Rho-associated protein kinase/MLCK pathway, establishing a cycle of barrier breakdown. Importantly, the present review highlights the lipocalin 10/slingshot homologue 1/cofilin axis and TJ-cytoskeleton interactions as mechanistic targets for future intervention in HAEC treatment. These findings provide a comprehensive mechanistic framework for understanding HAEC pathogenesis and offer novel targets for clinical intervention.

Organoids formed by combining pluripotent-stem-cell-derived human neural crest cells with pluripotent-stem-cell-derived intestinal tissue show functional interstitial cells of Cajal and undergo waves of contraction; these tissues reveal insights into the molecular defects characterizing Hirschsprung's disease. The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B . This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract.

Abstract Objectives Hirschsprung disease (HD) is a congenital condition defined by the absence of ganglion cells in the distal-most portion of the gastrointestinal tract. Biopsies and resections for HD can be adrenaline inducing for the general surgical pathologist because specimens are infrequent; HD is 1 of only a few neuroanatomic diseases that general surgical pathologists diagnose; numerous preanalytic factors (eg, biopsy adequacy, surgeon sampling protocol, processing artifacts) can affect histologic interpretation; and most importantly, the diagnosis has high stakes. Methods We provide a comprehensive overview of the background, relevant clinical procedures, and pathologic assessment of HD. Grossing and frozen section protocols, an algorithmic approach to diagnosis, and histologic pearls and pitfalls are also discussed. Results Evaluation and recognition of the features of HD have evolved significantly in the past 2 decades with the discovery of the value of calretinin immunohistochemistry in the late 2000s and the recent development of straightforward and reproducible histologic criteria for identification of the HD transition zone. Conclusions These advancements have substantially improved the pathologist’s ability to reliably evaluate for HD. Nonetheless, as with any high-stakes surgical pathology specimen, clear communication with the clinical team is essential.

PurposeHirschsprung's associated enterocolitis (HAEC) is a complication of Hirschsprung's Disease (HD) with considerable morbidity and mortality. The variability in presentation leads to a wide variety of the reported prevalence pre-and postoperatively. This systematic review aimed to clarify the prevalence of HAEC in short—(S-HD), long (L-HD), TCA and the type of operation used.MethodsA systematic literature-based search for relevant cohorts was performed using Pubmed/Medline, Cochrane Library from its inception to May 2021. Studies reporting on pre-and postoperative enterocolitis, segment length, and surgical procedure (Soave, Swenson, Duhamel) were included. Pooled prevalence and subgroup analysis have been calculated for pre-and postoperative HAEC.Results4738 articles were identified from the literature search, among which 57 studies, including 9744 preoperative and 8568 postoperative patients, were included. The groups were sorted by length of the aganglionic segment for further analysis. The pooled prevalence for preoperative HAEC was 18.3% for all types, 15.2% for S-HD and 26.1% for TCA. The pooled prevalence for postoperative HAEC was in total 18.2% for all segment lengths and used techniques. Subgroup analysis showed no significant difference in the occurrence of postoperative enterocolitis between the three techniques.ConclusionThe prevalence of preoperative HAEC increases with segment length. However, pooled data suggest that the postoperative risk for developing HAEC, independently of the employed method and segment length, is comparable to the preoperative risk.

Hirschsprung’s disease (HSCR) is a common cause of neonatal bowel obstruction and the approach to diagnosis and surgical treatment is well defined and accepted. Hirschsprung’s-associated enterocolitis (HAEC) remains a frequent cause of pre-operative and post-operative morbidity and mortality, with unchanged treatment guidelines over multiple decades. Recent advances in our understanding of the genetics underlying HSCR have allowed the development of animal models, some of which recapitulate the HAEC phenotype. These animal models, along with recent translational studies, have implicated multiple facets of mucosal immunity and microbiome dysbiosis in the development of HAEC. Here, we will review the established epidemiology, modes of diagnosis and treatment of HAEC. Furthermore, we will explore emerging concepts in the pathogenesis of this disease; including animal models, alterations in mucosal immunity, dysbiosis of the intestinal microbiome, specific genetic susceptibility, and novel treatment modalities.

A differentiation protocol to obtain enteric nervous system (ENS) progenitors and a range of neurons from human pluripotent stem cells is developed; the cells can migrate and graft to the colon of a chick embryo and an adult mouse colon, including in a mouse model of Hirschsprung disease, in which a functional rescue is observed. Enteric nervous system precursor cells The enteric nervous system (ENS) is essential to normal gut function and works to some extent independently from other neuronal circuits. Lorenz Studer and colleagues have developed a differentiation protocol for human pluripotent stem cells to obtain ENS progenitors and a range of neurons found in the ENS. They show that these can migrate and graft to the gut of a chick embryo and adult mouse colon, including in a mouse model of Hirschsprung disease, in which they observe a functional rescue. The authors also show that ENS precursors derived from patients affected by Hirschsprung disease can be used to screen for drugs that can modulate the properties of these cells. The enteric nervous system (ENS) is the largest component of the autonomic nervous system, with neuron numbers surpassing those present in the spinal cord 1 . The ENS has been called the ‘second brain’ 1 given its autonomy, remarkable neurotransmitter diversity and complex cytoarchitecture. Defects in ENS development are responsible for many human disorders including Hirschsprung disease (HSCR). HSCR is caused by the developmental failure of ENS progenitors to migrate into the gastrointestinal tract, particularly the distal colon 2 . Human ENS development remains poorly understood owing to the lack of an easily accessible model system. Here we demonstrate the efficient derivation and isolation of ENS progenitors from human pluripotent stem (PS) cells, and their further differentiation into functional enteric neurons. ENS precursors derived in vitro are capable of targeted migration in the developing chick embryo and extensive colonization of the adult mouse colon. The in vivo engraftment and migration of human PS-cell-derived ENS precursors rescue disease-related mortality in HSCR mice ( Ednrb s-l/s-l ), although the mechanism of action remains unclear. Finally, EDNRB-null mutant ENS precursors enable modelling of HSCR-related migration defects, and the identification of pepstatin A as a candidate therapeutic target. Our study establishes the first, to our knowledge, human PS-cell-based platform for the study of human ENS development, and presents cell- and drug-based strategies for the treatment of HSCR.

Hirschsprung-associated enterocolitis (HAEC) is one of the most severe complications in patients with Hirschsprung's disease (HSCR). Previous research has indicated that acetylcholine (ACH) plays an anti-inflammatory role during inflammation by acting on the α7 nicotinic acetylcholine receptor(α7nAchR) to promote the secretion of anti-inflammatory factors. However, the specific role of ACH in HAEC remains unclear. This experiment aims to explore the sources of ACH in HSCR and its anti-inflammatory mechanisms, thereby identifying new directions for the prevention and treatment of HAEC. We analyzed single-cell transcriptome data from HSCR to identify cells that secrete ACH and observed their distribution using immunofluorescence. In mice, F4/80, iNOS, ARG-1 and CD206 were used to identify and locate M1 and M2 macrophages in different intestinal segments. Western blot, reverse transcription-quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay were used to test the levels of IκBα, tumor necrosis factor-α, interleukin-10, and the macrophage activation pathway proteins JAK2 and STAT3 in different intestinal segments of mice. Organoid and cell culture techniques were used to verify the anti-inflammatory mechanism of ACH models. scRNA-seq analysis revealed that tuft cells expressed the CHAT protein. In HSCR, aganglionic segments exhibited heightened cholinergic activity compared with dilated ganglionic segments. In HAEC, inflammation was mainly concentrated in the dilated ganglionic segment and was associated with an increase in M1 macrophages, whereas the aganglionic segment showed less inflammation and was associated with an increase in M2 macrophages. Furthermore, experiments showed that intestinal organoids containing tuft cells promoted an increase in M2 macrophage markers, and ACH promoted M2 macrophage polarization. Differences in inflammation among various intestinal segments in HAEC may be linked to ACH secreted by tuft cells. Drugs targeting tuft cells have the potential to become important components of HAEC treatment in the future.

Background Advances in surgical techniques and perioperative care have improved the short- and mid-term postoperative outcomes of patients with Hirschsprung disease (HD). However, the long-term outcomes of these patients (older than 10 years) have not been fully investigated. The aim of this systematic review is to clarify the prevalence of long-term outcomes and the quality of life of these patients. Methods PubMed, AMED, Cochrane Library, CINAHL and PsycINFO databases were searched from inception to October 2018, following the Meta-analysis of Observational Studies in Epidemiology (MOOSE) guideline. Original studies reporting the outcomes of patients older than ten years with HD were selected and reviewed. The overall prevalence of fecal incontinence, constipation, bowel function score, bladder dysfunction symptoms, and patients’ quality of life were extracted from the included studies and pooled through the random-effects meta-analysis model. The heterogeneity and variation in the pooled estimations were evaluated by Cochrane’s Q test and the I 2 test. The sensitivity analysis was conducted by the sequential omission of individual studies. Publication bias was evaluated by Egger’s linear regression test. The whole procedure was conducted with Stata (version 14). Results In total, 3406 articles were identified from the literature search, among which twelve studies, including 625 patients, were included for analysis. The pooled prevalences of fecal incontinence, constipation, and bladder dysfunction symptoms and good to excellent bowel function scores were 0.20 (95% CI 0.13–0.28), 0.14 (95% CI 0.06–0.25), 0.07 (95% CI 0.04–0.12), and 0.95 (95% CI: 0.91–0.97), respectively; the pooled mean score of gastrointestinal-related quality of life was 118 (95% CI: 112.56–123.44). Conclusions HD patients older than ten years old have an overall high prevalence of fecal incontinence and a low quality of life. Targeted and evidence-based follow-up procedures and transitional care are essential to meet these patients’ long-term care needs. Prospective and multicenter research that focuses on the attributes and predictors of the long-term prognosis of patients with HD are necessary.

Hirschsprung disease (HSCR) is a neurocristopathy, yet paradoxically, neural crest-derived EGCs are present within the muscle of the affected region. This study investigates the molecular identity, origins, and neurogenic potential of EGCs in the aganglionic mouse and human colon. We utilized single-cell RNA sequencing (scRNA-seq), immunohistochemistry, and in vitro culture of EGCs from aganglionic and ganglionated segments of Ednrb-null mice ( Plp1-GFP;Baf-tdT;Ednrb −/− ) and human HSCR tissues. Neurogenic potential and network formation were assessed, and the effects of glial cell line-derived neurotrophic factor (GDNF) on neurogenesis were evaluated. scRNA-seq and immunohistochemistry revealed the absence of GFAP+ intraganglionic (IG) glia in aganglionic colon, while CAMK2b + extraganglionic (EG) glia and Schwann-like cells (SLCs) were present. EG glia exhibited a transcriptional profile similar to SLCs, suggesting a possible shared embryonic origin. EGCs in the aganglionic segment (comprising EGs and SLCs) exhibited reduced neurogenic potential and network complexity compared to EGCs from the ganglionated region (comprising EGs and IGs). GDNF partially restored neurogenic capacity and enhanced network complexity of EGCs isolated from the aganglionic segment, acting through a non-canonical NCAM1-dependent pathway independent of RET signaling.

Hirschsprung's disease (HSCR) is a severe congenital anomaly of the enteric nervous system (ENS) characterized by functional intestinal obstruction due to a lack of intrinsic innervation in the distal bowel. Distal innervation deficiency results from incomplete colonization of the bowel by enteric neural crest cells (eNCCs), the ENS precursors. Here, we report the generation of a mouse model for HSCR--named Holstein--that contains an untargeted transgenic insertion upstream of the collagen-6α4 (Col6a4) gene. This insertion induces eNCC-specific upregulation of Col6a4 expression that increases total collagen VI protein levels in the extracellular matrix (ECM) surrounding both the developing and the postnatal ENS. Increased collagen VI levels during development mainly result in slower migration of eNCCs. This appears to be due to the fact that collagen VI is a poor substratum for supporting eNCC migration and can even interfere with the migration-promoting effects of fibronectin. Importantly, for a majority of patients in a HSCR cohort, the myenteric ganglia from the ganglionated region are also specifically surrounded by abundant collagen VI microfibrils, an outcome accentuated by Down syndrome. Collectively, our data thus unveil a clinically relevant pathogenic mechanism for HSCR that involves cell-autonomous changes in ECM composition surrounding eNCCs. Moreover, as COL6A1 and COL6A2 are on human Chr.21q, this mechanism is highly relevant to the predisposition of patients with Down syndrome to HSCR.