Marcos Bettolli

Colonic dysmotility in postsurgical patients with Hirschsprung's disease. Potential significance of abnormalities in the interstitial cells of Cajal and the enteric nervous system

PURPOSE Normal gut muscular function depends on the coordinated activity of both the enteric nervous system (ENS) and the interstitial cells of Cajal (ICC). Hirschsprungs disease (HD) has long been considered a purely neuronal deficit but recent data point to abnormalities in ICC in the proximal ganglionated HD colon. We examined the labeling of ICC and neuronal cells in the proximal ganglionated colon in patients with HD to determine whether abnormalities of ICC and ENS might be associated with a poor clinical outcome. METHODS Tissue from 11 patients with HD was studied using immunohistochemistry for ICC and neuronal identification in comparison to control tissue from patients without HD. Image data were evaluated quantitatively and interpreted relative to clinical outcome. RESULTS Interstitial cells of Cajal in the ganglionated colon of the HD group did not differ from the control group, but nerve cells/fibers were decreased 40%. Paired decreases in both nerve fibers and ICC in individual patients were associated with normal bowel function. Poor postoperative outcome was observed in a patient with normal innervation but with a profound decrease in ICC in the ganglionated colon. CONCLUSIONS Nerve fibers are decreased in the proximal ganglionated colon in patients with HD without associated gut dysmotility. Poor clinical outcome was noted only in a patient with normal innervation and markedly decreased ICC. Collection of data from a much larger number of patients with poor clinical outcome will be necessary to determine the significance of this imbalance of ICC and innervation.

Gene expression profiling in necrotizing enterocolitis reveals pathways common to those reported in Crohn's disease

BackgroundNecrotizing enterocolitis (NEC) is the most frequent life-threatening gastrointestinal disease experienced by premature infants in neonatal intensive care units. The challenge for neonatologists is to detect early clinical manifestations of NEC. One strategy would be to identify specific markers that could be used as early diagnostic tools to identify preterm infants most at risk of developing NEC or in the event of a diagnostic dilemma of suspected disease. As a first step in this direction, we sought to determine the specific gene expression profile of NEC.MethodsDeep sequencing (RNA-Seq) was used to establish the gene expression profiles in ileal samples obtained from preterm infants diagnosed with NEC and non-NEC conditions. Data were analyzed with Ingenuity Pathway Analysis and ToppCluster softwares.ResultsData analysis indicated that the most significant functional pathways over-represented in NEC neonates were associated with immune functions, such as altered T and B cell signaling, B cell development, and the role of pattern recognition receptors for bacteria and viruses. Among the genes that were strongly modulated in neonates with NEC, we observed a significant degree of similarity when compared with those reported in Crohn’s disease, a chronic inflammatory bowel disease.ConclusionsGene expression profile analysis revealed a predominantly altered immune response in the intestine of NEC neonates. Moreover, comparative analysis between NEC and Crohn’s disease gene expression repertoires revealed a surprisingly high degree of similarity between these two conditions suggesting a new avenue for identifying NEC biomarkers.

Interstitial cell of Cajal loss correlates with the degree of inflammation in the human appendix and reverses after inflammation.

BACKGROUND Normal gut motility relies on the complex interaction between the interstitial cell of Cajal (ICC) and the enteric nerve networks. Inflammation of the gastrointestinal tract adversely affects both ICC and enteric nerves. We aimed to determine the distribution of ICC and nerve networks in patients with appendicitis. METHODS Specimens from controls and patients with appendicitis were examined with immunohistochemistry (c-Kit for ICC, beta III tubulin [Tuj-1] and neuronal nitric oxide synthase [histochemical diaphorase] for nitrergic neurons) and electron microscopy (EM). Data were quantified using image analysis. RESULTS We found a profound decrease in c-Kit immunoreactivity (c-Kit IR) in the advanced inflammatory stages of appendicitis, which correlated with the severity of inflammation. Electron microscopy confirmed ultrastructural injury in both ICC and nerve fiber networks during acute inflammation. After the inflammation resolved, interval appendices displayed a recovery in ICC c-Kit IR to control levels and normal ultrastructure. The neuronal network also displayed ultrastructural recovery; however, neuronal nitric oxide synthase activity did not recover. CONCLUSIONS Severe inflammation results in significant ultrastructural damage of nerves and ICC networks in appendicitis. The loss of c-Kit IR is likely due to impaired ICC cytophysiology because ICC was still present under EM. After resolution of acute inflammation, ICC recovers their normal ultrastructure and c-Kit IR.

Laparoscopic-Assisted Colostomy in Children

INTRODUCTION Colostomy morbidity has been reported to be as high as 50%. Laparoscopic-assisted colostomy (LAC) is associated with decreased colostomy complication. LAC is recommended for stoma formation in adults but has not been previously reported in children. In this paper, we report on our initial experience with LAC in children. MATERIALS AND METHODS Using a two- to four-port (3.5-mm) technique, LAC was performed in a female with an imperforate anus and 2 male patients with complicated Hirschsprungs disease (HD), respectively. Data collected included operative time, time to recover bowel function, and morbidity. Close follow-up was done until stoma closure. RESULTS The operative time was 144 minutes in the HD patients (including concomitant laparoscopic biopsies and a leveling colostomy) and 40 minutes in the imperforate anus patient. Median time to passage of both flatus and stool was 40 hours (range, 24-48). Time to commence feeds postop was 40 hours (range, 24-48). The median time of follow-up was 3 months (range, 2-9) until the stoma was taken down. No complications have occurred to date. CONCLUSIONS LAC is safe and easily performed in neonates and infants. It facilitates accurate stoma placement and orientation. It allows additional bowel mobilization, especially in HD. In accordance with the adult experience, LAC seems to obviate stoma-related complications. Encouraged by our initial low morbidity rate, a prospective evaluation of this technique is planned.

A less intensive bracing protocol for pectus carinatum

OBJECTIVES Despite the widespread use of bracing to correct Pectus carinatum (PC) there is no consensus in the number of hours per day patients are instructed to wear the brace. In our practice, we use a less rigorous protocol of 8-12h/day. We sought to evaluate our results and those in the literature to determine whether more intensive usage is necessary. STUDY DESIGN We reviewed the outcomes of patients with PC treated at our institution between 2012 and 2015. We searched MEDLINE, EMBASE and Web of Science for studies describing the use of bracing to correct PC. RESULTS Seventy-five patients presented with PC at our institution. Among those who were offered bracing and had adequate follow-up (n=32), the success rate (full correction or improvement) was 90.6%. The compliance rate was 93.8%. Fifteen studies met our inclusion criteria. Our pooled data combining our results with those of other published data showed that less intensive brace usage (<12h/day), when compared to more intensive usage (≥12h/day), is associated with higher patient compliance (89.6% vs. 81.1%) with a similar time to correction (7.3 vs 7.1months) and success rate (85.3% vs. 83.5%). CONCLUSIONS Implementing a less intensive bracing protocol for PC is successful, efficient and improves compliance. TYPE OF STUDY Clinical Research. LEVELS OF EVIDENCE Oxford Centre for Evidence-Based Medicine Level-of-Evidence rating: Level IV.

Growth-Associated Protein 43 Expression in Ganglionic and Aganglionic Colon

Abstract Objectives: Because of its specificity for nerve fibers of the enteric nervous system, calretinin is an effective adjunctive marker in the assessment for Hirchsprung disease. Growth associated protein (GAP-43) has been shown to be expressed in nerve fibers within the intestinal lamina propria. No prior report compares GAP-43 expression in ganglionic versus aganglionic intestine. Methods: Six consecutive Hirschsprung endorectal pull through specimens were retrieved from our archives. In addition 3 controls were selected from colonic resections for reasons other than Hirschsprung Disease. Immunoperoxidase for GAP-43 was carried out on the ganglionic and aganglionic segments of all cases and controls. Submucosal ganglion soma positivity and nerve fiber positivity within the lamina propria were graded on a subjective scale of 1-3 that incorporated both strength and density. Data: GAP-43 strongly stained submucosal ganglion cells and nerve fibers within the lamina propria in 6/6 of the ganglionic segments and 3/3 of the normally innervated controls . GAP-43 did not show any ganglion cell body positivity within the aganglionic segments; however, all 6 aganglionic segment lamina propria were positive for nerve fiber staining. There was a small subjective increase in the amount of nerve fiber positivity for GAP-43 in ganglionic segments and controls versus aganglionic segments. Conclusion: GAP-43 marks mucosal nerve fibers in ganglionic intestine but also aganglionic intestine and thus is less useful than calretinin as a marker for Hirschsprung Disease. The abundant mucosal nerves highlighted by GAP-43 requires further characterization.

A unique presentation of a standard type C esophageal atresia in a very low-birth-weight neonate

Neonates with esophageal atresia and tracheoesophageal fistula usually present with inability to swallow immediately after birth often associated with respiratory distress. This is an unusual presentation of a very low-birth-weight neonate with a type C tracheoesophageal fistula that was fed for the first 4 days of life through an unintentional tracheogastric tube without incident.

Impaired antimicrobial response and mucosal protection induced by ibuprofen in the immature human intestine

BackgroundThe use of nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin (INDO) and ibuprofen (IBU) has been shown to be an effective therapy for the closure of patent ductus arteriosus (PDA). However, this treatment has been associated with an increased risk of developing enteropathies in neonates. Whether the use of IBU is safer than INDO for the immature intestine remains to be elucidated.MethodsThe direct impact of IBU on the human immature intestinal transcriptome was investigated using serum-free organ culture. Differentially expressed genes were analyzed with Ingenuity Pathway Analysis software and compared with those previously reported with INDO. Validation of differentially expressed genes was confirmed by qPCR.ResultsWe identified several biological processes that were significantly modulated by IBU at similar levels to what had previously been observed with INDO, while the expression of genes involved in “antimicrobial response” and “mucus production” was significantly decreased exclusively by IBU in the immature intestine.ConclusionsOur findings indicate that IBU has a harmful influence on the immature intestine. In addition to exerting many of the INDO observed deleterious effects, IBU alters pathways regulating microbial colonization and intestinal epithelial defense.

Vacuum Bell Treatment of Pectus Excavatum: an Early North American Experience

PURPOSE Conservative treatment of pectus excavatum with a vacuum bell device may be an attractive alternative to surgical repair. We describe an early North American experience with this device. METHODS Prospectively maintained chest wall clinic registries from two institutions were reviewed to identify pectus excavatum patients ≤21 years treated with the vacuum bell from 2013 to 2017. Multivariate linear regression was used to compare mean improvements in deformity-depth and Haller Index between groups of patients based on age and usage metrics (hours/day and days/week). RESULTS Thirty-one patients with a median age of 14 years received treatment with the device. Mean follow-up duration was 18 months. Median depth and Haller Index at treatment onset were 2.3 cm and 3.9, respectively. Improvements in deformity-depth were superior with device usage >2 h/day (p < 0.01) and daily use (p < 0.01). After adjusting for compliance, younger age of treatment onset was associated with greater improvement in Haller Index but not deformity depth. CONCLUSION Our prospective early North American experience found the vacuum bell to be a potential alternative to surgical treatment for pectus excavatum. Longer usage periods in a daily frequency are associated with best results. TYPE OF STUDY Treatment study; case series with no comparison group. LEVEL OF EVIDENCE Level IV.