The Effect of the Ganglionic Segment Inflammatory Response to Postoperative Enterocolitis in Hirschsprung Disease.

INTRODUCTION: We examined the relationship between proinflammatory cytokines that occur in the inflammatory reaction in the intestine in Hirschsprung disease (HD) and Hirschsprung-associated enterocolitis (HAEC). METHODS: Thirty cases (M:27, F:3) operated on due to HD. The cases were divided into three groups: group 1 with pre and post operative EC, group 2 with post-operative, and group 3 with pre-operative EC. The intestinal segments were evaluated by immunohistochemistry for interleukin 1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6). RESULTS: IL-1ß staining was significantly higher in the ganglionic zone of groups with enterocolitis compared to the control group (p = 0.012). TNF-α staining in the transitional zone of Group 3 and IL-1ß staining in the ganglionic zone of Group 1 was significantly higher than the control group (p = 0.030, p = 0.020). CONCLUSION: In our study, older age at diagnosis and more than 20% IL-1ß staining in the ganglionic segment were found to be risk factors for HAEC. It is noteworthy that the increase in IL-1ß can be associated with HAEC.

Histological transitional zone pull-through in Hirschsprung disease. Postoperative functional results and current recommendations.

BACKGROUND: Surgeons create a neorectum to repair patients with Hirschsprung's disease (HD), which should be formed from a normoganglionic bowel. However, the neorectum is occasionally created with a transition zone (TZ) bowel. A neorectum created with a TZ has been postulated as a cause of postoperative enterocolitis or constipation. This study compares the incidence of enterocolitis and constipation in patients with TZ neorectum and normoganglionic bowel. METHODS: We conducted a retrospective review of patients with rectosigmoid HD who underwent primary pull-through. Patients were divided into normoganglionic neorectum (NNR) and TZ neorectum. The diagnosis was based on the final histopathologic report of the proximal margin. The incidence of enterocolitis and constipation was compared between these two groups. RESULTS: A total of 98 HD patients were analyzed. Seventy-one patients fulfilled the inclusion criteria. 65 (92%) had a NNR, and six patients (8%) had a TZ neorectum. From these patients, 42 (59%) presented with enterocolitis or constipation. However, there was no significant difference between both groups. CONCLUSION: The present study showed no difference in the incidence of enterocolitis or postoperative constipation in HD patients with normoganglionic or TZ neorectum. These results suggest that TZ neorectum does not cause postoperative obstructive symptoms.

INTRODUCCIÓN: Los cirujanos crean un neo-recto para tratar a los pacientes con enfermedad de Hirschsprung (EH), que debe formarse con intestino normogangliónico; sin embargo, en ocasiones el neo-recto se forma con intestino de la zona de transición. Se ha postulado que un neo-recto en zona de transición causa enterocolitis o estreñimiento postoperatorio. El objetivo de este estudio fue comparar la frecuencia de enterocolitis y estreñimiento en pacientes con neo-recto en zona de transición y con neo-recto normogangliónico. MÉTODOS: Se llevó a cabo una revisión retrospectiva de pacientes con EH recto sigmoideo que se sometieron a descenso primario. Los pacientes se dividieron en el grupo neo-recto normogangliónico y el grupo con neo-recto en zona de transición. El diagnóstico del neo-recto se estableció con el informe histopatológico definitivo del margen proximal. Se comparó la frecuencia de enterocolitis y estreñimiento entre estos dos grupos. RESULTADOS: Se analizó un total de 98 pacientes con EH, de los cuales 71 pacientes cumplieron los criterios de inclusión; 65 (92%) con neo-recto normogangliónico y seis (8%) con neo-recto en zona de transición. Posteriormente, 42 (59%) pacientes presentaron enterocolitis asociada a Hirschsprung (HAEC) o estreñimiento; sin embargo, no hubo diferencia significativa entre ambos grupos. CONCLUSIONES: El presente estudio no demostró una diferencia en la frecuencia de HAEC o estreñimiento postoperatorio en pacientes con EH con neo-recto normogangliónico o en zona de transición. Estos resultados sugieren que un neo-recto en zona de transición no causa síntomas obstructivos postoperatorios.

Suction rectal biopsy (SRB) in Hirschsprung's Disease: Is a single macroscopically adequate sample sufficient?

Hirschsprung's Disease (HD) is a congenital disorder causing severe constipation in infants and children. Suction rectal biopsy (SRB) is the preferred technique for obtaining tissue samples for histopathological evaluation. In low-resource settings like Malaysia, cost-effective diagnostic approaches are necessary, making single sample SRB valuable. This study evaluates the diagnostic accuracy and sufficiency of a single macroscopically adequate sample in suction rectal biopsies for the histopathological confirmation of HD. We conducted a retrospective study of children who underwent suction rectal biopsies for the diagnosis of HD at Hospital Raja Perempuan Zainab II (HRPZII), Kota Bharu, Kelantan. A total of 68 patients were included in the study. The inadequacy rate for bedside SRB was 14%, comparable to current literature. Our study found no statistically significant association between sample inadequacy and gestational age, gender, birth weight, or weight at biopsy. Complication rates were 0%, consistent with literature reports. Calretinin staining, an additional technique, was performed in 23 biopsy episodes, with a 4.3% inadequacy rate, compared to 20% in specimens not subjected to calretinin staining. The cost of SRB almost doubled with each additional sample taken, significant in low-resource environments. In conclusion, single sample SRBs can be adequately diagnostic and cost-effective in low-resource settings, providing valuable insights for healthcare facilities in Malaysia and other developing countries. The use of adjunctive techniques such as calretinin staining may improve diagnostic accuracy while maintaining cost-effectiveness. Further prospective studies with larger sample sizes are needed to validate these findings.

A novel mouse model of intestinal neuronal dysplasia: visualization of the enteric nervous system.

PURPOSE: Intestinal neuronal dysplasia (IND) is a congenital anomaly affecting gastrointestinal neural innervation, but the pathogenesis remains unclear. The homozygous Ncx/Hox11L.1 knockout (Ncx-/-) mice exhibit megacolon and enteric ganglia anomalies, resembling IND phenotypes. Sox10-Venus transgenic mouse were used to visualize enteric neural crest cells in real time. This study aims to establish a novel mouse model of Sox10-Venus+/Ncx-/- mouse to study the pathogenesis of IND. METHODS: Sox10-Venus+/Ncx-/- (Ncx-/-) (n = 8) mice and Sox10-Venus+/Ncx+/+ controls (control) (n = 8) were euthanized at 4-5 weeks old, and excised intestines were examined with fluorescence microscopy. Immunohistochemistry was performed on tissue sections with neural marker Tuj1. RESULTS: Ncx-/- mice exhibited dilated cecum and small intestine. Body weight of Ncx-/- mice was lower with higher ratio of small intestine length relative to body weight. The neural network (Sox10-Venus) was observed along the intestine wall in Ncx-/- and control mice without staining. Ectopic and increased expression of Tuj1 was observed in both small intestine and proximal colon of Ncx-/- mice. CONCLUSION: This study has established a reliable animal model that exhibits characteristics similar to patients with IND. This novel mouse model can allow the easy visualization of ENS in a time- and cost-effective way to study the pathogenesis of IND.

Hirschsprung disease.

Hirschsprung disease (HSCR) is a rare congenital intestinal disease that occurs in 1 in 5,000 live births. HSCR is characterized by the absence of ganglion cells in the myenteric and submucosal plexuses of the intestine. Most patients present during the neonatal period with the first meconium passage delayed beyond 24 h, abdominal distension and vomiting. Syndromes associated with HSCR include trisomy 21, Mowat-Wilson syndrome, congenital central hypoventilation syndrome, Shah-Waardenburg syndrome and cartilage-hair hypoplasia. Multiple putative genes are involved in familial and isolated HSCR, of which the most common are the RET proto-oncogene and EDNRB. Diagnosis consists of visualization of a transition zone on contrast enema and confirmation via rectal biopsy. HSCR is typically managed by surgical removal of the aganglionic bowel and reconstruction of the intestinal tract by connecting the normally innervated bowel down to the anus while preserving normal sphincter function. Several procedures, namely Swenson, Soave and Duhamel procedures, can be undertaken and may include a laparoscopically assisted approach. Short-term and long-term comorbidities include persistent obstructive symptoms, enterocolitis and soiling. Continued research and innovation to better understand disease mechanisms holds promise for developing novel techniques for diagnosis and therapy, and improving outcomes in patients.

A unicentric cross-sectional observational study on chronic intestinal inflammation in total colonic aganglionosis: beware of an underestimated condition.

BACKGROUND: Inflammatory Bowel Diseases (IBD) are known to occur in association with Hirschsprung disease (HSCR). Most of cases are represented by Crohn Disease (CD) occurring in patients with Total Colonic Aganglionosis (TCSA) with an estimated prevalence of around 2%. Based on these considerations and on a number of provisional data belonging to our Center for Digestive Diseases, we developed a unicentric cross-sectional observational study aimed at describing phenotype, genotype, pathology and metagenomics of all patients with TCSA and Crohn-like lesions. RESULTS: Out of a series of 62 eligible TCSA patients, 48 fulfilled inclusion criteria and were enrolled in the study. Ten patients did not complete the study due to non-compliance or withdrawal of consent and were subsequently dropped out. A total of 38 patients completed the study. All patients were tested for chronic intestinal inflammation by a combination of fecal calprotectine (FC) or occult fecal blood (OFB) and underwent fecal metagenomics. Nineteen (50%) tested positive for FC, OFB, or both and subsequently underwent retrograde ileoscopy. Fourteen patients (36.8%) presented Crohn-like lesions, occurring after a median of 11.5 years after surgery (range 8 months - 21.5 years). No statistically significant differences regarding demographic, phenotype and genotype were observed comparing patients with and without lesions, except for need for blood transfusion that was more frequent in those with lesions. Faecal microbiome of patients with lesions (not that of caregivers) was less biodiverse and characterized by a reduction of Bacteroidetes, and an overabundance of Proteobacteria. FC tested negative in 3/14 patients with lesions (21%). CONCLUSIONS: Our study demonstrated an impressive 10-folds higher incidence of chronic inflammation in TCSA. Up to 50% of patients may develop IBD-like lesions postoperatively. Nonetheless, we failed in identifying specific risk factors to be used to implement prevention strategies. Based on the results of our study, we suggest screening all TCSA patients with retrograde ileoscopy regardless of FC/OFB values. The frequency of endoscopic assessments and the role of FC/OFB screening in prompting endoscopy is yet to be determined.

Suppression of PGE2/EP2 signaling alleviates Hirschsprung disease by upregulating p38 mitogen-activated protein kinase activity.

Hirschsprung disease (HSCR) is a congenital disorder caused by the failure of enteric neural crest cells (ENCCs) to colonize the distal bowel, resulting in absence of enteric nervous system. While a range of molecules and signaling pathways have been found to contribute to HSCR development, the risk factors and pathogenesis of this disease in many patients remain unknown. We previously demonstrated that increased activity of the prostaglandin E2 (PGE2)/PGE2 receptor subtype EP2 pathway can be a risk factor for HSCR. In this study, an Ednrb-deficient mouse model of HSCR was generated and used to investigate if PGE2/EP2 pathway could be a potential therapeutic target for HSCR. We found that downregulation of PGE2/EP2 signaling by siRNA-mediated ablation of a PGE2 synthase or pharmacologic blockage of EP2 enhanced ENCC colonization in the distal bowel of Ednrb-/- mice and alleviated their HSCR-like symptoms. Furthermore, blockage of EP2 was shown to promote ENCC migration through upregulating p38 mitogen-activated protein kinase activity, which was downregulated in the colon of Ednrb-/- mice and in the distal aganglionic bowel of HSCR patients. These data provide evidence that maternal exposure during embryonic development to an environment with dysregulated activation of the PGE2/EP2 pathway may predispose genetically susceptible offspring to HSCR, and avoidance or early disruption of maternal events (e.g. inflammation) that possibly enhance PGE2/EP2 signaling during pregnancy would reduce the occurrence and severity of this disease. KEY MESSAGES : Knockdown of PTGES alleviates HSCR severity in Ednrb-/- mice. Blockage of EP2-mediated PGE2 signaling alleviates HSCR severity in Ednrb-/- mice. Blockage of EP2-mediated PGE2 signaling promotes ENCC migration via enhancing p38 activity.

Systematic orientation of fresh rectal suction biopsies improves histopathological diagnostics in hirschsprung's disease - a method description and preliminary report.

BACKGROUND: Optimizing rectal suction biopsy (RSB) diagnostics in Hirschsprung's disease (HD) may shorten diagnostic time and prevent need for repeated biopsies. AIM: To explore whether systematic orientation of fresh RSB specimens increased biopsy quality, diagnostic times, diagnostic efficacy, and histopathologic workload, and to explore these outcome measures for aganglionic specimens. MATERIALS/METHODS: This was an observational case-control study conducted at a national referral center for HD on data collected from the local HD-diagnostic register. From 2019 each fresh RSB was oriented by the collector in a notch in a foam cushion, placed in a separate cassette, and sent in formalin for pathological analysis. Outcome measures of oriented RSB samples collected 2019-2021 were compared to those of non-oriented RSB samples collected 2015-2018. Staining/immunohistochemistry consisted of hematoxylin eosin, S-100 and calretinin. RESULTS: 78 children with 81 RSBs and 242 biopsy analyzes were included. The frequency of high-quality RSB specimens was higher in oriented: 40% (42/106) versus non-oriented 25% (34/136) (p = 0.018), the diagnostic turnaround time was shorter: 2 days (1-5) versus 3 days (2-8) (p = 0.015), and the number of additional sectioning/leveling/re-orientation per biopsy was lower: 7 (3-26) versus 16 (7-72) (p = 0.011). Specifically for aganglionic specimens, the frequency of high-quality biopsies was generally higher in oriented than in non-oriented RSB specimens: 47% (28/59) versus 14% (7/50) (p < 0.001); the diagnostic efficacy was higher 95% (19/20) versus 60% (9/15) (p = 0.027) and the diagnostic turnaround time shorter: 2 days (2-3) versus 3 days (2-8) (p = 0.036). CONCLUSIONS: Systematic orientation of fresh RSB specimens improves HD diagnostics. Improvement was consistent in aganglionic specimens.

Development of the aganglionic colon following surgical rescue in a cell therapy model of Hirschsprung disease in rat.

Patients with Hirschsprung disease lack enteric ganglia in the distal colon and propulsion of colorectal content is substantially impaired. Proposed stem cell therapies to replace neurons require surgical bypass of the aganglionic bowel during re-colonization, but there is inadequate knowledge of the consequences of bypass. We performed bypass surgery in Ednrb-/- Hirschsprung rat pups. Surgically rescued rats failed to thrive, an outcome reversed by supplying electrolyte- and glucose-enriched drinking water. Histologically, the bypassed colon had normal structure, but grew substantially less in diameter than the functional region proximal to the bypass. Extrinsic sympathetic and spinal afferent neurons projected to their normal targets, including arteries and the circular muscle, in aganglionic regions. However, although axons of intrinsic excitatory and inhibitory neurons grew into the aganglionic region, their normally dense innervation of circular muscle was not restored. Large nerve trunks that contained tyrosine hydroxylase (TH)-, calcitonin gene-related peptide (CGRP, encoded by Calca or Calcb)-, neuronal nitric oxide synthase (nNOS or NOS1)-, vasoactive intestinal peptide (VIP)- and tachykinin (encoded by Tac1)-immunoreactive axons occurred in the distal aganglionic region. We conclude that the rescued Ednrb-/- rat provides a good model for the development of cell therapies for the treatment of Hirschsprung disease.

A new systematization of histological analysis for the diagnosis of Hirschsprung's disease.

BACKGROUND: Hirschsprung's Disease (HD) is characterized by intestinal sub-occlusion and the absence of enteric ganglion cells. A rectal biopsy examination is performed to confirm the diagnosis. In a recent study, we demonstrated that the analysis of 60 sections of rectal mucosa and submucosa stained by H&E may ensure a 90% diagnostic accuracy. Although the need to analyze so many sections makes the process of reading the slides more time-consuming, this encouraged us to study their distribution in the healthy rectal submucosa, to simplify the diagnosis. OBJECTIVES: To develop a method that facilitates HD diagnosis by studying the distribution of ganglion cells in the submucosal plexus. METHODS: Using the calretinin technique, we studied the distribution of plexuses in 60 fragments of rectal submucosa from 19 cadavers. After the study, the reading method created was used for diagnosis in 47 cases of suspected HD, using H&E staining. The accuracy was verified by comparing the results obtained with H&E to those obtained with the acetylcholinesterase technique, the golden standard in our laboratory. RESULTS: The study of submucosal plexus distribution showed that just by examining the submucosal region every 20 µm, approximately, it is possible to locate a ganglionic plexus, and we have already been able to diagnose HD with 93% accuracy. CONCLUSION: The study of ganglion cell distribution enabled the creation of a simplified method for reading the slides. The method applied achieved good accuracy and it can be used as an alternative method in HD diagnosis.

Tissue Shrinkage of Resected Specimens in Hirschsprung's Disease: Why Pediatric Surgeons Think the Bowel Specimen was Longer Than Indicated in the Pathology Report.

BACKGROUND: Hirschsprung disease (HD) is an aganglionosis of variable length starting at the rectosigmoid colon with surgery as sole therapeutic option. The length of the resected bowel segment is a crucial information for the treating surgeons and influences the prognosis of the patient. It is often artificially altered due to post operative tissue shrinkage. The objective of this study is to quantify the extent tissue shrinkage of HD specimens. MATERIAL AND METHODS: Colorectal HD specimens were measured at the time of surgery and at the time of cut-up, either fresh or after formalin fixation and statistically analyzed. RESULTS: Sixteen colorectal specimens were included. Following formalin fixation the specimen length decreased by 22.7% (P < .001). Without formalin fixation the specimens shrank by an average of 24.9% (P = .05). There was no significant difference in the extent of tissue shrinkage with or without formalin fixation (P = .76). CONCLUSION: This study showed that there is significant tissue shrinkage in HD specimens. The 2 different cohorts revealed that tissue shrinkage is mostly caused by tissue retraction/alteration after organ removal but also to a lesser extent by fixation with formalin. Surgeons and (neuro-)pathologists should be aware of the sizeable shrinking artifact to avoid unnecessary confusion.

Diagnosing Hirschsprung Disease in Children Younger than 6 Months of Age: Insights in Incidence of Complications of Rectal Suction Biopsy and Other Final Diagnoses.

BACKGROUND: The gold standard for diagnosing Hirschsprung disease (HD) in patients younger than 6 months is pathological examination of rectal suction biopsy (RSB). The aim of this study was to gain insight into the following: (1) complications following RSB, (2) final diagnosis of patients referred for RSB, and (3) factors associated with HD. METHODS: Patients suspected of HD referred for RSB at our center were analyzed retrospectively. Severity of complications of RSB was assessed using Clavien-Dindo (CD) grading. Factors associated with HD were tested using multivariate logistic regression analysis. RESULTS: From 2000 to 2021, 371 patients underwent RSB because of infrequent defecation, at a median age of 44 days. Three patients developed ongoing rectal bleeding (0.8%) graded CD1. Most frequent final diagnoses were: HD (n = 151, 40.7%), functional constipation (n = 113, 31%), idiopathic meconium ileus (n = 11, 3%), and food intolerance (n = 11, 3%). Associated factors for HD were male sex (odds ratio [OR], 3.19; confidence interval [CI], 1.56-6.53), presence of syndrome (OR, 7.18; CI, 1.63-31.69), younger age at time of RSB (OR, 0.98; CI, 0.85-0.98), meconium passage for more than 48 hours (OR, 3.15; CI, 1.51-6.56), distended abdomen (OR, 2.09; CI, 1.07-4.07), bilious vomiting (OR, 6.39; CI, 3.28-12.47), and failure to thrive (OR, 8.46; CI, 2.11-34.02) (model R 2 = 0.566). CONCLUSION: RSB is a safe procedure with few and only minor complications. In the majority of patients referred for RSB under the age of 6 months, HD was found followed by a functional cause for the defecation problems. RSB should be obtained on a low threshold in all patients under the age of 6 months with the suspicion of HD.

Study on the application of optical coherence microscopy in Hirschsprung's disease.

To explore the clinical application value of optical coherence microscopy (OCM) in Hirschsprung's disease. 109 HSCR patients were recuited in a Chinese hospital from January 2018 to July 2021. All the recruited patients underwent barium enema angiography preoperatively and the resected diseased intestinal tubes were evaluated intraoperatively. The OCM and the histopathological examination were performed successively on the surgical specimens, and the OCM images were compared with the relevant tissue sections to characterize different lesions. 10 non-HSCR fetal colorectal tissues at the same period were retained for OCM, the characteristics of which with and without HSCR under OCM imaging were analyzed. In the OCM images of in vitro tissue, it can be clearly observed that the scattering degree of HSCR narrow segment mucosal is high, glands and crypt structures are reduced or even atrophy, and the scattering degree of submucosal and intermuscular is low; In the dilated segment, the low scattering and high scattering are complex, and the muscle layer is obviously hypertrophy and structural disorder. Compared with the pathological findings, the OCM sensitivity, Kappa value, and AUC area reached 92.66%, 0.63, and 0.91, respectively. OCM can quickly and clearly display the structure of all layers of colorectal tissue, which is highly consistent with the corresponding histopathological examination results and has high sensitivity. which will provide a more reliable basis for OCM diagnosis of early HSCR, targeted biopsy and location of operative treatment, and has a certain potential for clinical application.

Immunologic Heterogeneity in 2 Cartilage-Hair Hypoplasia Patients With a Distinct Clinical Course.

BACKGROUND AND OBJECTIVE: Cartilage-hair hypoplasia (CHH) syndrome is a rare autosomal recessive syndrome associated with skeletal dysplasia, varying degrees of combined immunodeficiency (CID), short stature, hair hypoplasia, macrocytic anemia, increased risk of malignancies, and Hirschsprung disease. To provide clinical and immunological insights obtained from 2 unrelated patients who displayed clinical characteristics of CHH. METHODS: Two patients with suspected CHH syndrome due to skeletal dysplasia and immunodeficiency underwent an immunological and genetic work-up using flow cytometry, next-generation sequencing (NGS) of the immune repertoire, and Sanger sequencing to identify the underlying defects. RESULTS: Patient 1 presented with low birth weight and skeletal dysplasia. Newborn screening was suggestive of T-cell immunodeficiency, as T-cell receptor excision circle levels were undetectable. Both the T-cell receptor (TCR) Vß and TCR-g (TRG) repertoires were restricted, with evidence of clonal expansion. Genetic analysis identified compound heterozygous RMRP variants inherited from both parents. Patient 2 presented with recurrent lung and gastrointestinal infections, skeletal dysplasia, failure to thrive, and hepatomegaly. The polyclonal pattern of the TCRß repertoire was normal, with only slight overexpression of TCR-ßV20 and restricted expression of Vßs. TRG expressed a normal diverse repertoire, similar to that of the healthy control sample. Genetic analysis identified biallelic novel regulatory variants in RMRP. Both parents are carriers of this mutation. CONCLUSION: Our findings demonstrate how the immunological work-up, supported by genetic findings, can dramatically change treatment and future outcome in patients with the same clinical syndrome.

Hirschsprung Disease for the Practicing Surgical Pathologist.

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.

Reoperation for Hirschsprung Disease: Two cases of Vanishing Ganglion Cells and Review of the Literature.

Hirschsprung disease (HD) is characterized by circumferential aganglionosis of the rectum with variable proximal bowel involvement. The underlying pathogenesis is due to failure of caudal migration of neural crest cells during pre-natal development, causing functional bowel obstruction. Definitive therapy is surgical resection; however, a subset of patients will require reoperation. An important cause of reoperation is the rare but distinct entity described as the ganglion cell "vanishing" phenomenon. In this phenomenon, affected patients have normal circumferential ganglion cells present at the proximal margin during primary resection. They undergo a variable asymptomatic period post-primary resection but ultimately develop recurrent symptoms. Upon reoperation, ganglion cells seemingly vanish and are no longer present in the previously functioning and ganglionated bowel proximal to the initial anastomotic site. To further characterize and investigate this poorly understood pathology, here we present 2 cases of HD patients who required reoperation. Our small series implicates that an immune component may contribute as patient 2 had a brisk neurotrophic eosinophilic infiltrate only present in the reoperation specimen. However, this was not observed in patient 1. Other possible etiologies include post-operative ischemia/hypoxia, visceral neuropathy, or signaling abnormalities within the residual ganglion cells themselves.

Immunologic Heterogeneity in 2 Cartilage-Hair Hypoplasia Patients With a Distinct Clinical Course

Introduction: Cartilage-hair hypoplasia (CHH) syndrome is a rare autosomal recessive syndrome associated with skeletal dysplasia, varying degrees of combined immunodeficiency (CID), short stature, hair hypoplasia, macrocytic anemia, increased risk of malignancies, and Hirschsprung disease. Purpose: To provide clinical and immunological insights obtained from 2 unrelated patients who displayed clinical characteristics of CHH. Methods: Two patients with suspected CHH syndrome due to skeletal dysplasia and immunodeficiency underwent an immunological and genetic work-up using flow cytometry, next-generation sequencing (NGS) of the immune repertoire, and Sanger sequencing to identify the underlying defects. Results: Patient 1 presented with low birth weight and skeletal dysplasia. Newborn screening was suggestive of T-cell immunodeficiency, as T-cell receptor excision circle levels were undetectable. Both the T-cell receptor (TCR) Vß and TCR-g (TRG) repertoires were restricted, with evidence of clonal expansion. Genetic analysis identified compound heterozygous RMRP variants inherited from both parents. Patient 2 presented with recurrent lung and gastrointestinal infections, skeletal dysplasia, failure to thrive, and hepatomegaly. The polyclonal pattern of the TCRß repertoire was normal, with only slight overexpression of TCR-ßV20 and restricted expression of Vßs. TRG expressed a normal diverse repertoire, similar to that of the healthy control sample. Genetic analysis identified biallelic novel regulatory variants in RMRP. Both parents are carriers of this mutation. Conclusion: Our findings demonstrate how the immunological work-up, supported by genetic findings, can dramatically change treatment and future outcome in patients with the same clinical syndrome (AU)

Children with Hirschsprung disease in a developing country: A cohort study of the predictors of a positive rectal biopsy result.

Chronic constipation, which may be habitually or pathologically caused, is one of the most common complaints in children. One of the important pathological causes is Hirschsprung's disease (HD), which is diagnosed via multiple modalities, mainly rectal biopsy. Our aim was to compare the presentation and different predictive factors for positive rectal biopsy results in a developing country in the Middle East, such as Jordan. This cohort study was conducted at the Jordan University Hospital (JUH). All consecutive children aged <14 years who presented with refractory constipation and underwent rectal biopsies between January 2014 and December 2019 were retrospectively enrolled in the study. In the entire cohort study, 79 patients were enrolled: 45 (57%) were males and 34 (43%) were females. Regarding the biopsy results, 51 (64.6%) cases of refractory constipation without HD and 28 (35.4%) patients with refractory constipation with HD were diagnosed with open rectal biopsies. The male-to-female ratio of HD patients was 3:1. Moreover, 3 (10.7%) children who passed the meconium within the first 24 to 48 hours showed features of HD, while 17 (60.7%) children with delayed passage of the meconium showed features of HD. Abdominal distension was found to be a positive predictor of positive biopsy results (odds ratio [OR] = 4.09, P = .011), and soiling was found to be a negative predictor of positive biopsy results (OR = 0.07, P = .024). In developing countries, children presenting with HD seem to have similar symptoms and signs to those observed with traditional sampling and staining techniques.

Calretinin Staining in Anorectal Line Biopsies Accurately Distinguished Hirschsprung Disease in a Retrospective Study.

INTRODUCTION: The absence of submucosal ganglion cells does not reliably distinguish Hirschsprung disease from non Hirschsprung disease in anorectal line biopsies. Calretinin staining might be helpful in these biopsies. To determine its value, we analyzed calretinin positive mucosal neurites in anorectal line biopsies. METHODS: Two pediatric pathologists, without access to patient data, evaluated calretinin positive mucosal neurites in anorectal line junctional mucosa in archival rectal biopsies contributed by 17 institutions. A separate investigator compiled patient information and sent data for statistical analysis. RESULTS: Biopsies with anorectal junctional mucosa from 115 patients were evaluated for calretinin positive mucosal neurites. 20/20 Hirschsprung disease biopsies were negative. 87/88 non Hirschsprung disease biopsies and 7/7 post pullthrough Hirschsprung disease neorectal biopsies were positive. Statistical analysis of the 108 non pullthrough biopsies yielded an accuracy of 99.1% (sensitivity 100%, specificity 98.9%). Age range was preterm to 16 years. Biopsy size was less than 1 mm to over 1 cm. CONCLUSIONS: Absence of calretinin positive mucosal neurites at the anorectal line was highly accurate in distinguishing Hirschsprung disease from non Hirschsprung disease cases in this blinded retrospective study. Calretinin staining is useful for interpreting biopsies from the physiologic hypoganglionic zone up to the anorectal line.

miR­92a represses the viability and migration of nerve cells in Hirschsprung's disease by regulating the KLF4/PI3K/AKT pathway.

Hirschsprung's disease (HSCR) is an intestinal disease caused by defects in neural crest cell migration, proliferation, differentiation, and survival. Many reports have proposed that miRNA dysregulation is related to the occurrence of HSCR. However, the roles and mechanisms of miRNAs have not been thoroughly studied. The levels of miR­92a and KLF4 were examined using qRT­PCR and immunohistochemistry, respectively. Cell viability, migration and apoptosis were evaluated by MTT, Transwell and flow cytometry assays, respectively. A dual­luciferase reporter assay was employed to verify the binding relationship between miR­92a and KLF4. Levels of PI3K/AKT signals were further determined by western blot assay. Herein, elevated expression of miR­92a and reduced expression of KLF4 were found in HSCR tissues, and their expression patterns were negatively correlated. Overexpression of miR­92a inhibited cell viability and migration but enhanced cell apoptosis. However, overexpression of KLF4 had the opposite effects. Mechanistically, KLF4 was a target of miR­92a and it negatively affected biological functions by activating PI3K/AKT signaling. These results proved that miR­92a inhibited the proliferation and metastasis of nerve cells by regulating the KLF4/PI3K/AKT axis.