Reduction of hydrogen sulfide synthesis enzymes cystathionine-ß-synthase and cystathionine-γ-lyase in the colon of patients with Hirschsprungs disease.

PURPOSE: Hirschsprung associated enterocolitis (HAEC) is the most common cause of morbidity and mortality in Hirschsprung Disease (HSCR). The pathogenesis of HAEC is poorly understood. In recent years, there is increasing evidence that a compromised intestinal barrier function plays a major role in the pathogenesis of HAEC. Hydrogen sulfide, synthesized from L-cysteine by two key enzymes, cystathionine-ß-synthase (CBS) and cystathionine-γ-lysase (CSE) is reported to play a key role in regulating gastrointestinal motility and promoting resolution of inflammation. We designed this study to test the hypothesis that CBS and CSE expression is altered in the colon of patients with HSCR. METHODS: We investigated CBS and CSE protein expression in both the aganglionic and ganglionic regions of HSCR patients (n=10) versus healthy control colon (n=10). Protein distribution was assessed by using immunofluorescence and confocal microscopy. Gene and protein expression was quantified using quantitative real-time polymerase chain reaction (qPCR), Western blot analysis, and densitometry. MAIN RESULTS: qPCR and Western blot analysis revealed that CBS and CSE are expressed in the normal human colon. CBS and CSE expression was significantly decreased (p<0.003) in the ganglionic and aganglionic bowel in HSCR compared to controls. Confocal microscopy revealed that CBS and CSE expression in smooth muscles, interstitial cells of Cajal, platelet-derived growth factor-alpha receptor-positive cells, enteric neurons and colonic epithelium was markedly decreased in HSCR specimens compared to controls. CONCLUSION: We demonstrate for the first time the expression and distribution of CBS/CSE in patients with HSCR. The observed decreased expression of CBS and CSE may affect mucosal integrity and colonic contractility and thus render HSCR patients more susceptible to develop HAEC.

Human Erythrocyte Acetylcholinesterase in Health and Disease.

The biochemical properties of erythrocyte or human red blood cell (RBC) membrane acetylcholinesterase (AChE) and its applications on laboratory class and on research are reviewed. Evidence of the biochemical and the pathophysiological properties like the association between the RBC AChE enzyme activity and the clinical and biophysical parameters implicated in several diseases are overviewed, and the achievement of RBC AChE as a biomarker and as a prognostic factor are presented. Beyond its function as an enzyme, a special focus is highlighted in this review for a new function of the RBC AChE, namely a component of the signal transduction pathway of nitric oxide.

Modified Rapid AChE Method (MRAM) for Hirschsprung Disease Diagnosis: A Journey from Meier-Ruge Until Now.

Hirschsprung disease (HSCR) can be diagnosed using a variety of histological and immunohistochemical methods and stains. Because of the nature of the condition and the need for a rapid diagnostic confirmation, those methods with high accuracy and fast turnaround times are preferred. The authors of this paper have used rapid acetylcholinesterase (AChE) immunohistochemistry in conjunction with standard H&E in order to optimize diagnostic accuracy, and present a modified rapid AChE method (MRAM) that has been successfully utilized for over 20 years. The authors also present a list of over 30 different methods and stains that have been proposed for Hirschsprung disease diagnosis.

Altered tryptophan hydroxylase 2 expression in enteric serotonergic nerves in Hirschsprung's-associated enterocolitis.

AIM: To determine if expression of colonic tryptophan hydroxylase-2 (TPH2), a surrogate marker of neuronal 5-hydroxytryptamine, is altered in Hirschsprung's-associated enterocolitis. METHODS: Entire resected colonic specimens were collected at the time of pull-through operation in children with Hirschsprung's disease (HSCR, n = 12). Five of these patients had a history of pre-operative Hirschsprung's-associated enterocolitis (HAEC). Controls were collected at colostomy closure in children with anorectal malformation (n = 10). The distribution of expression of TPH2 was evaluated using immunofluorescence and confocal microscopy. Protein expression of TPH2 was quantified using western blot analysis in the deep smooth muscle layers. RESULTS: TPH2 was co-expressed in nitrergic and cholinergic ganglia in the myenteric and submucosal plexuses in ganglionic colon in HSCR and healthy controls. Co-expression was also seen in submucosal interstitial cells of Cajal and PDGFRα(+) cells. The density of TPH2 immuno-positive fibers decreased incrementally from ganglionic bowel to transition zone bowel to aganglionic bowel in the myenteric plexus. Expression of TPH2 was reduced in ganglionic bowel in those affected by pre-operative HAEC compared to those without HAEC and healthy controls. However, expression of TPH2 was similar or high compared to controls in the colons of children who had undergone diverting colostomy for medically refractory HAEC. CONCLUSION: Altered TPH2 expression in colonic serotonergic nerves of patients with HSCR complicated by HAEC may contribute to intestinal secretory and motor disturbances, including recurrent HAEC.

Associations Between CYP2B6 rs707265, rs1042389, rs2054675, and Hirschsprung Disease in a Chinese Population.

BACKGROUND: Previous studies suggested that cytochrome P450 participated in the tumor metastasis and migration. CYP2B6 also acts as an important enzyme which metabolize partially or primarily metabolism of drugs, environmental contaminants, and mutagens. The objective of this study was to investigate the influence of CYP2B6 polymorphism on susceptibility of Hirschsprung disease. METHODS: TaqMan assay was performed to determine the genotypes of CYP2B6 rs707265, rs1042389, rs2054675 in 262 cases and 290 control subjects. Logistic regression was used to assess the associations between these polymorphisms and HSCR. RESULTS: We observed a significant association of CYP2B6 rs707265 (G>A) polymorphism and HSCR susceptibility (p < 0.001). Besides, rs707265 A presented a significant risk of HSCR (p < 0.001). CONCLUSIONS: Our result suggested that CYP2B6 rs707265 modified the risk of HSCR.

Application of acetylcholinesterase histochemistry for the diagnosis of Hirschsprung's disease in neonates and infants: a twenty-year experience.

BACKGROUND: The acetylcholinesterase (AChE) histochemical staining of intestinal mucosal-submucosal biopsy specimens is believed to be the most reliable diagnostic method for Hirschsprung's disease (HD). The aim of our study was to evaluate advantages and disadvantages of this method for HD diagnosis in infants and neonates. MATERIAL AND METHODS: The results of AChE histochemistry of rectal biopsy specimens, obtained from 11 neonates and 29 infants treated in the Clinic of Pediatric Surgery, Hospital of the Lithuanian University of Health Sciences, from 1991 to 2010 were analyzed. AChE activity of neural structures was evaluated using Karnovsky-Roots method modified by El-Badawi and Schenk. RESULTS: Two neonates were diagnosed with HD. The diagnosis was not confirmed in 9 cases, but clinical symptoms progressed in 3 cases, and HD was diagnosed after the repeated biopsy performed in infancy. The results of primary biopsy were rated as false negative. Test sensitivity and specificity in neonates were 40.0% and 100%, respectively. A total of 21 infants were diagnosed with HD. All of them underwent surgery. The diagnosis of HD was confirmed in 20 cases; in one case, intestinal neuronal dysplasia type B was diagnosed. The diagnosis was not confirmed in 8 cases. In infants, the test had a sensitivity of 100% and a specificity of 88.8%. CONCLUSIONS: The analysis of AChE activity in rectal biopsy specimens is a reliable method for diagnosing HD in infancy. This test is less valuable in neonates. If test results are negative, infants should be observed, and if symptoms persist, the biopsy should be repeated at the age of 3 months. Rectal biopsy specimens in neonates should include mucosa and submucosa.

The involvement of nitric oxide synthase neurons in enteric neuropathies.

Nitric oxide (NO), produced by the neural nitric oxide synthase enzyme (nNOS) is a transmitter of inhibitory neurons supplying the muscle of the gastrointestinal tract. Transmission from these neurons is necessary for sphincter relaxation that allows the passage of gut contents, and also for relaxation of muscle during propulsive activity in the colon. There are deficiencies of transmission from NOS neurons to the lower esophageal sphincter in esophageal achalasia, to the pyloric sphincter in hypertrophic pyloric stenosis and to the internal anal sphincter in colonic achalasia. Deficits in NOS neurons are observed in two disorders in which colonic propulsion fails, Hirschsprung's disease and Chagas' disease. In addition, damage to NOS neurons occurs when there is stress to cells, in diabetes, resulting in gastroparesis, and following ischemia and reperfusion. A number of factors may contribute to the propensity of NOS neurons to be involved in enteric neuropathies. One of these is the failure of the neurons to maintain Ca(2+) homeostasis. In neurons in general, stress can increase cytoplasmic Ca(2+), causing a Ca(2+) toxicity. NOS neurons face the additional problem that NOS is activated by Ca(2+). This is hypothesized to produce an excess of NO, whose free radical properties can cause cell damage, which is exacerbated by peroxynitrite formed when NO reacts with oxygen free radicals.

Correlating of GSTM1, GSTT1, and GSTP1 genetic polymorphisms with the risk and expressions in children with isolated Hirschsprung disease.

PURPOSE: The present study aimed to examine an association between the glutathione S-transferases (GSTs) polymorphisms (GSTM1, GSTT1, and GSTP1) genetic polymorphisms with the risk and expression in children with isolated Hirschsprung disease (HD). METHODS: GSTM1, GSTT1, and GSTP1 genetic polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism analysis in 80 HD and 180 normal children (controls). The genic expressions were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The protein expressions were detected by Western blot. RESULTS: The GSTM1 null genotype especially is associated with a greater risk of HD (X(2) = 1.129, P = 0.288, OR = 0.851, 95% CI = 0.632-1.146). The GSTT1 null genotype especially is associated with a greater risk of HD (X(2) = 6.165, P = 0.013, OR = 1.472, 95% CI = 1.084-1.999). The GSTP1 null genotype especially is associated with a greater risk of HD (X(2) = 4.748, P = 0.029, OR = 0.701, 95% CI = 0.509-0.964). GSTP1 and GSTP1 expressions were higher than GSTM1 in HD patients. Positive expressive rate of the GSTT1 and GSTP1 were 40.56% and 56.67% in HD. The mRNA and protein expressions level of GSTT1 and GSTP1 genes were significantly higher in HD than controls (P < 0.05). Positive expressive rate of the GSTM1 was 10.56% in HD. The GSTM1 was low expressed between in HD and controls (P > 0.05). CONCLUSIONS: The GSTP1 genetic polymorphisms correlate to HD. We postulate that inherited gene deletion of GSTT1 and GSTP1 may produce increased genotoxic susceptibility for HD respectively, following exposure to xenobiotics that are substrates for these enzymes.

An infant with cartilage-hair hypoplasia due to a novel homozygous mutation in the promoter region of the RMRP gene associated with chondrodysplasia and severe immunodeficiency.

Cartilage-hair hypoplasia (CHH) is a rare autosomal-recessive disorder characterized by short-limbed dwarfism, sparse hair, and immune deficiency. It is caused by mutations in the RMRP gene, which encodes the RNA component of the mitochondrial RNA-processing ribonuclease (RNase MRP). Several mutations have been identified in its promoter region or transcribed sequence. However, homozygous mutations in the promoter region have been only reported in a patient with primary immunodeficiency without other features of CHH. We report on a Thai girl who first presented with chronic diarrhea, recurrent pneumonia, and severe failure to thrive, without apparently disproportionate dwarfism. The diagnosis of CHH was made after the severe wasting was corrected, and disproportionate growth became noticeable. The patient had the typical features of CHH, including sparse hair and metaphyseal abnormalities. The immunologic profiles were consistent with combined immune deficiency. Mutation analysis identified a novel homozygous mutation, g.-19_-25 dupACTACTC, in the promoter region of the RMRP gene. Identification of the mutation enabled us to provide a prenatal diagnosis in the subsequent pregnancy. This patient is the first CHH case with the characteristic features due to the homozygous mutation in the promoter region of the RMRP gene. The finding of severe immunodeficiency supports that promoter mutations markedly disrupt mRNA cleavage function, which causes cell-cycle impairment.

c-Ret-mediated hearing loss in mice with Hirschsprung disease.

A significantly increased risk for dominant sensorineural deafness in patients who have Hirschsprung disease (HSCR) caused by endothelin receptor type B and SOX10 has been reported. Despite the fact that c-RET is the most frequent causal gene of HSCR, it has not been determined whether impairments of c-Ret and c-RET cause congenital deafness in mice and humans. Here, we show that impaired phosphorylation of c-Ret at tyrosine 1062 causes HSCR-linked syndromic congenital deafness in c-Ret knockin (KI) mice. The deafness involves neurodegeneration of spiral ganglion neurons (SGNs) with not only impaired phosphorylation of Akt and NF-kappaB but decreased expression of calbindin D28k in inner ears. The congenital deafness involving neurodegeneration of SGNs in c-Ret KI mice was rescued by introducing constitutively activated RET. Taken together with our results for three patients with congenital deafness with c-RET-mediated severe HSCR, our results indicate that c-Ret and c-RET are a deafness-related molecule in mice and humans.

Analyses of PRMT1 proteins in human colon tissues from Hirschsprung disease patients.

BACKGROUND: Protein arginine methyltransferase 1 (PRMT1) catalyzes the majority of arginine methylation in cells and plays important roles in the differentiation and development of neurons. It is also implicated in the regulation of nitric oxide synthetase (NOS). Hirschsprung disease (HSCR) is characterized by the absence of intramural ganglion cells in the nerve plexuses of the distal gut. METHODS: Western blot analyses revealed reduced PRMT1 protein levels in the aganglionosis segments of HSCR patients. Immunohistochemistry detected PRMT1 expression in the colonic mucosa, the enteric nervous system (ENS) and endothelial cells. Specific and strong PRMT1 expression in neuron cell bodies of the plexus was demonstrated by immunofluorescent double-labeling with neuron-specific marker HuC/D. KEY RESULTS: In the mucosa, PRMT1 was detected at all crypt cells. Intensive PRMT1 staining was detected in the submucosal and the myenteric plexuses in normal or oligoganglionosis segments. Aganglionosis segments from HSCR patients contain no plexuses, and thus not labeled with PRMT1. The phenomenon is specific to the megacolon of HSCR as strong PRMT1 staining was observed in plexuses of the rectal ectasia segments (dilated rectum and distal sigmoid not related with aganglionosis) from anorectal malformation patients. Furthermore, PRMT1 was also present in the same neuronal cells expressing neuronal NOS in the plexuses. CONCLUSIONS & INFERENCES: We suggest that PRMT1 can be a useful marker for HSCR. This study is the first illustration of PRMT1 protein expression in human tissues from non-cancerous disease and set up the base for further investigations of PRMT1 function in ENS development and intestinal motility.

Neural precursor death is central to the pathogenesis of intestinal aganglionosis in Ret hypomorphic mice.

The RET tyrosine kinase is required for the migration, proliferation, and survival of the enteric neural crest-derived cells (ENCCs) that form the enteric nervous system (ENS). Hypomorphic RET alleles cause intestinal aganglionosis [Hirschsprung disease (HSCR)], in which delayed migration and successive nonapoptotic ENCC death are considered to be major contributory factors. The significance of ENCC death in intestinal aganglionosis, however, has remained unclear. We show that elevated expression of Bcl-xL inhibits ENCC death in both Ret-null and hypomorphic states. However, the rescued Ret-null mice showed ENS malfunction with reduced nitric oxide synthase expression in colonic neurons, revealing the requirement of RET for neuronal differentiation. In contrast, the inhibition of cell death allows morphologically and functionally normal ENS formation in Ret hypomorphic mice. These results indicate that ENCC death is a principal cause of intestinal aganglionosis in a Ret hypomorphic state, and suggest that the inhibition of cell death is a route to the prevention of HSCR.

Reduced RET expression in gut tissue of individuals carrying risk alleles of Hirschsprung's disease.

Receptor tyrosine kinase (RET) single nucleotide polymorphisms (SNPs) are associated with the Hirschsprung's disease (HSCR). We investigated whether the amount of RET expressed in the ganglionic gut of human was dependent on the genotype of three regulatory SNPs (-5G>A rs10900296 and -1A>C rs10900297 in the promoter, and C>T rs2435357 in intron 1). We examined the effects of three regulatory SNPs on the RET gene expression in 67 human ganglionic gut tissues using quantitative real-time PCR. Also, 315 Chinese HSCR patients and 325 ethnically matched controls were genotyped for the three SNPs by polymerase chain reaction (PCR) and direct sequencing. The expression of RET mRNA in human gut tissue did indeed correlate with the genotypes of the individuals. The lowest RET expression was found for those individuals homozygous for the three risk alleles (A-C-T/A-C-T), and the highest for those homozygous for the 'wild-type' counterpart (G-A-C/G-A-C), with expression values ranging from 218.32 +/- 125.69 (mean +/- SE) in tissues from individuals carrying G-A-C/G-A-C to 31.42 +/- 8.42 for individuals carrying A-C-T/A-C-T (P = 0.018). As expected, alleles -5A, -1C and intron 1 T were associated with HSCR (P = 5.94 x 10(-31), 3.12 x 10(-24) and 5.94 x 10(-37), respectively) as was the haplotype encompassing the three associated alleles (A-C-T) when compared with the wild-type counterpart G-A-C (chi2 = 155.29, P << 0.0001). To our knowledge, this is the first RET expression genotype-phenotype correlation study conducted on human subjects to indicate common genetic variants in the regulatory region of RET may play a role in mediating susceptibility to HSCR, by conferring a significant reduction of the RET expression.

Activation of MAP kinase (ERK1/2) in human neonatal colonic enteric nervous system.

The aim of this study was to examine mitogen-activated protein kinase (ERK1/2) activation in the human neonatal colonic enteric nervous system. For this, we investigated by immunocytochemistry the cellular localization of phosphorylated ERK1/2 (P-ERK) in a series of normal human colon samples removed from newborns and in patients with intestinal obstruction such as Hirschsprung's disease (HSCR), stenosis and atresia. We checked the presence of P-ERK in the three distinct histological layers of normal colon. Phosphorylated ERK was detected in the colonic mucosa, in the enteric nervous system and in endothelial cells. In the mucosa from normal colon, P-ERK was detected at the upper part of the crypt, while P-ERK activation in epithelial cells is altered in HSCR, stenosis and atresia. In the normal colon, strong P-ERK staining was detected in myenteric and submucosal enteric plexuses. Using confocal microscopy analyses, we observed that P-ERK staining was localized in enteric glial cells and not in enteric neurons. Strong P-ERK staining was also observed in plexuses from stenosis and atresia whereas in HSCR, hypertrophic nerve fibres were not stained.

Diagnosis and surgical treatment of isolated hypoganglionosis.

BACKGROUND: Some patients suspected with Hirschsprung's disease (HD), however, were diagnosed as having isolated hypoganglionosis according to the updated pathohistologic methods. This study was undertaken to investigate the diagnostic methods and the therapeutic results of isolated hypoganglionosis in children. METHODS: A retrospective analysis was made on 17 patients with isolated hypoganglionosis (hypoganglionosis group) identified pathologically after operation. The data included clinical presentations, barium enema, anorectal manometry, histochemical staining for acetylcholinesterase (AChE) before operation, histological results after operation and follow-up outcomes. The data of hypoganglionosis with HD (HD group) were compared retrospectively. RESULTS: Common complaint of the patients with hypoganglionosis and HD was intractable constipation. Barium enema showed typical narrowing and distended segment of the colon in 9 patients in the hypoganglionosis group (9/16) and in 15 patients in the HD group (15/18). In the hypoganglionosis group, in 15 patients who underwent anorectal manometry only 5 showed absent rectal anal inhibitory reflex, significantly lower than the rate in the HD group (17/18) (P<0.05). From 16 patients in hypoganglionosis group, positive staining for AChE was noted in 3 patients (3/16, 18.8%), significantly lower than that in the HD group (16/18, 88.9%) (P<0.05). Thirteen patients in the hypoganglionosis group received subtotal colectomy, while only 5 patients needed subtotal colectomy in the HD group. In the hypoganglionosis group, except 2 patients who suffered from mild enterocolitis after operation and recovered after conservative therapy, all patients recovered uneventfully without wound dehiscence, intestinal fistula, fecal incontinence or constipation recurrence. In the HD group, one patient suffered from anastomotic leak and got secondary operation, one patient had anastomotic stricture at 1 year after operation and recovered by dilatation, and other three patients suffered from mild enterocolitis after operation and recovered after conservative therapy. CONCLUSIONS: Hypoganglionosis is a common disease, and could be finally confirmed by full-thickness biopsies in different bowel segments. The resection range can be estimated according to barium enema and 24-hour delayed X-ray findings, by which the satisfactory result in short-term follow-up can be obtained.

Role of rectal biopsy in predicting response to intrasphincteric botulinum toxin injection for obstructive symptoms after a pullthrough operation.

OBJECTIVE: Our aim was to correlate the pathological results and clinical response in patients who underwent botulinum toxin (BT) injection for obstructive symptoms (OS) after a pullthrough operation for Hirschsprung's disease (HD). METHODS: Between August 2002 and February 2006, 16 of 107 HD patients (15%) were referred with persistent OS after pull-through (PT) operation in this center. They underwent rectal biopsy and BT injection in the internal sphincter. Their responses to BT injection were evaluated by the constipation score before, and at 1, 3 and 8 months after the injection, and anorectal manometry (ARM) before and at 2 weeks, and 1 and 8 months after the injection. The association between response to BT and acetylcholinesterase (AChE) staining of rectal biopsy was also assessed. RESULTS: Fourteen of 16 patients (87%) had improvement in bowel function after 2 weeks, and two patients did not respond at all. Six of the 14 patients with early response had recurrence of symptoms after 2-3 months. Eight patients with normal ganglia and negative AChE had good response with no recurrence on follow-up. However, 4 of 6 recurrences were neurogenic dysfunctions and 2 were intestinal neuronal dysplasia (2-4+AChE). Two patients with no response had an aganglionic segment (4+AChE). Four of 6 patients with recurrence showed improvement with BT re-injection and only 2 did not improve. CONCLUSION: A higher degree of AChE staining is associated with lack of response to BT injection. This is also a test for predicting the severity of neurogenic dysfunction in the intestinal wall.

Studies of RET gene expression and acetylcholinesterase activity in a series of sporadic Hirschsprung's disease.

The purpose is to present the studies of RET gene expression and acetylcholinesterase activity in 23 patients operated for Hirschsprung's disease (HD). The patients underwent either transanal endorectal pull-through or Duhamell's procedure. Full-thickness intestinal samples from the three different segments (ganglionic, intermediate and aganglionic) were collected. Each tissue sample was divided in two portions, one for AChE histochemical staining and the other for examination of RET mRNA expression level. All patients had an uneventful postoperative recovery. In all patients, the AChE stainings demonstrated the absence of activity in the ganglionic area, the marked increase of positive fibers in the aganglionic area, and little increase of positive fibers in the intermediate area. In the ganglionic and intermediate areas, all patients (100%) showed significant RET gene expression. In the aganglionic area, 18 patients (78.3%) did not present gene expression and the other five patients (21.7%) presented gene expression that was similar to the ganglionic and intermediate areas. The results reinforce the conclusion that the method of AChE staining is effective for the diagnosis of intestinal aganglionosis and confirm the knowledge that genes beyond RET may be implicated in the genesis of sporadic cases of HD.

Variability of acetylcholinesterase hyperinnervation patterns in distal rectal suction biopsy specimens in Hirschsprung disease.

Variability in cholinergic innervation may contribute to false-negative and false-positive evaluations for Hirschsprung disease (HD). We compared intraspecimen variability of histochemical acetylcholinesterase (ACE) activity in 62 distal rectal mucosal biopsy specimens from 33 patients with short-segment HD (SSHD), 14 patients with short-segment HD/Down syndrome (SSHD/DS), 10 patients with total colonic aganglionosis (HDTC), and 45 ganglionated control specimens obtained because of clinical suspicion for HD. Specimens were evaluated in 1-mm linear segments for abnormal distribution and abnormal intensity of staining reaction in the lamina propria (LP), muscularis mucosae (MM), numerical excess of small submucosal nerves (SSN), and presence or absence of large submucosal nerves (LSN) defined as >35 microns in diameter. Patients with HD and controls were predominantly infant males. Aggregate length of specimens was 186 mm in HD and 136 mm in controls. We defined multiple patterns and relative frequencies of normal and abnormal ACE reactivity in the muscularis mucosae, submucosal nerves, and innervation in the lamina propria. We verified a hyperinnervation pattern more common in neonates within each subset of HD. Large submucosal nerves >35 microns in diameter are relatively less common in SSHD/DS and HDTC. Eleven of 57 patients with HD had at least 1 mm of normal muscularis mucosae accounting for 19/186 mm examined. Ten percent of control mm had at least 1 submucosal nerve >35 microns in diameter. Intraspecimen variability in ACE patterns, when extreme, can usually be resolved by findings elsewhere in an adequate specimen, minimizing the need for repeat procedures.

Uncommon causes of postoperative chronic diarrhoea mimicking enterocolitis in Hirschsprung's disease: is there a role for digestive endoscopy?

Severe chronic diarrhoea secondary to enterocolitis is a severe complication of Hirschsprung's disease (HSCR). Persistent outlet obstruction, immunologic issues, and mucin/mucous imbalance can cooperate in the development of this complication. Furthermore, isolated reports described severe postoperative chronic diarrhoea mimicking enterocolitis in patients with sucrase-isomaltase deficiency, inflammatory bowel disease (IBD), or intestinal microvillus atrophy. This paper is aimed in describing three patients from our HSCR series who experienced severe chronic postoperative diarrhoea secondary to such uncommon associated anomalies: sucrase-isomaltase deficiency (one patient) and IBD (two patients). With an appropriate sucrose-free diet or immunosuppressive therapy these patients improved dramatically and their diarrhoea settled. These associated anomalies can be diagnosed with digestive endoscopies (both gastro-duodenoscopy and colonoscopy). Therefore, we developed a diagnostic and therapeutic algorithm for patients with chronic diarrhoea after a pull-through, which includes digestive endoscopy to be performed in selected cases.