Patient-derived enteroids provide a platform for the development of therapeutic approaches in microvillus inclusion disease.

Microvillus inclusion disease (MVID), caused by loss-of-function mutations in the motor protein myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid/base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na+/H+ exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking antidiarrheal drug crofelemer dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. γ-Secretase inhibition with DAPT recovered apical brush border structure and functional Na+/H+ exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum/glucocorticoid-regulated kinase 2 (SGK2) and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID.

Therapy Development for Microvillus Inclusion Disease using Patient-derived Enteroids.

Microvillus Inclusion Disease (MVID), caused by loss-of-function mutations in the motor protein Myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid-base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex Immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na + /H + exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking anti-diarrheal drug, Crofelemer, dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. Inhibition of Notch signaling with the γ-secretase inhibitor, DAPT, recovered apical brush border structure and functional Na + /H + exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum- and glucocorticoid-induced protein kinase 2 (SGK2), and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID. Conflict-of-interest statement: The authors have declared that no conflict of interest exists.

Editing Myosin VB Gene to Create Porcine Model of Microvillus Inclusion Disease, With Microvillus-Lined Inclusions and Alterations in Sodium Transporters.

BACKGROUND & AIMS: Microvillus inclusion disease (MVID) is caused by inactivating mutations in the myosin VB gene (MYO5B). MVID is a complex disorder characterized by chronic, watery, life-threatening diarrhea that usually begins in the first hours to days of life. We developed a large animal model of MVID to better understand its pathophysiology. METHODS: Pigs were cloned by transfer of chromatin from swine primary fetal fibroblasts, which were edited with TALENs and single-strand oligonucleotide to introduce a P663-L663 substitution in the endogenous swine MYO5B (corresponding to the P660L mutation in human MYO5B, associated with MVID) to fertilized oocytes. We analyzed duodenal tissues from patients with MVID (with the MYO5B P660L mutation) and without (controls), and from pigs using immunohistochemistry. Enteroids were generated from pigs with MYO5B(P663L) and without the substitution (control pigs). RESULTS: Duodenal tissues from patients with MVID lacked MYO5B at the base of the apical membrane of intestinal cells; instead MYO5B was intracellular. Intestinal tissues and derived enteroids from MYO5B(P663L) piglets had reduced apical levels and diffuse subapical levels of sodium hydrogen exchanger 3 and SGLT1, which regulate transport of sodium, glucose, and water, compared with tissues from control piglets. However, intestinal tissues and derived enteroids from MYO5B(P663L) piglets maintained CFTR on apical membranes, like tissues from control pigs. Liver tissues from MYO5B(P663L) piglets had alterations in bile salt export pump, a transporter that facilitates bile flow, which is normally expressed in the bile canaliculi in the liver. CONCLUSIONS: We developed a large animal model of MVID that has many features of the human disease. Studies of this model could provide information about the functions of MYO5B and MVID pathogenesis, and might lead to new treatments.

Loss of MYO5B Leads to Reductions in Na+ Absorption With Maintenance of CFTR-Dependent Cl- Secretion in Enterocytes.

BACKGROUND & AIMS: Inactivating mutations in MYO5B cause microvillus inclusion disease (MVID), but the physiological cause of the diarrhea associated with this disease is unclear. We investigated whether loss of MYO5B results in aberrant expression of apical enterocyte transporters. METHODS: We studied alterations in apical membrane transporters in MYO5B-knockout mice, as well as mice with tamoxifen-inducible, intestine-specific disruption of Myo5b (VilCreERT2;Myo5bflox/flox mice) or those not given tamoxifen (controls). Intestinal tissues were collected from mice and analyzed by immunostaining, immunoelectron microscopy, or cultured enteroids were derived. Functions of brush border transporters in intestinal mucosa were measured in Ussing chambers. We obtained duodenal biopsy specimens from individuals with MVID and individuals without MVID (controls) and compared transporter distribution by immunocytochemistry. RESULTS: Compared to intestinal tissues from littermate controls, intestinal tissues from MYO5B-knockout mice had decreased apical localization of SLC9A3 (also called NHE3), SLC5A1 (also called SGLT1), aquaporin (AQP) 7, and sucrase isomaltase, and subapical localization of intestinal alkaline phosphatase and CDC42. However, CFTR was present on apical membranes of enterocytes from MYO5B knockout and control mice. Intestinal biopsies from patients with MVID had subapical localization of NHE3, SGLT1, and AQP7, but maintained apical CFTR. After tamoxifen administration, VilCreERT2;Myo5bflox/flox mice lost apical NHE3, SGLT1, DRA, and AQP7, similar to germline MYO5B knockout mice. Intestinal tissues from VilCreERT2;Myo5bflox/flox mice had increased CFTR in crypts and CFTR localized to the apical membranes of enterocytes. Intestinal mucosa from VilCreERT2;Myo5bflox/flox mice given tamoxifen did not have an intestinal barrier defect, based on Ussing chamber analysis, but did have decreased SGLT1 activity and increased CFTR activity. CONCLUSIONS: Although trafficking of many apical transporters is regulated by MYO5B, trafficking of CFTR is largely independent of MYO5B. Decreased apical localization of NHE3, SGLT1, DRA, and AQP7 might be responsible for dysfunctional water absorption in enterocytes of patients with MVID. Maintenance of apical CFTR might exacerbate water loss by active secretion of chloride into the intestinal lumen.

Apical Membrane Alterations in Non-intestinal Organs in Microvillus Inclusion Disease.

OBJECTIVES: Microvillus inclusion disease (MVID) is a severe form of neonatal diarrhea, caused mainly by mutations in MYO5B. Inactivating mutations in MYO5B causes depolarization of enterocytes in the small intestine, which gives rise to chronic, unremitting secretory diarrhea. While the pathology of the small intestine in MVID patients is well described, little is known about extraintestinal effects of MYO5B mutation. METHODS: We examined stomach, liver, pancreas, colon, and kidney in Navajo MVID patients, who share a single homozygous MYO5B-P660L (1979C>T p.Pro660Leu, exon 16). Sections were stained for markers of the apical membrane to assess polarized trafficking. RESULTS: Navajo MVID patients showed notable changes in H/K-ATPase-containing tubulovesicle structure in the stomach parietal cells. Colonic mucosa was morphologically normal, but did show losses in apical ezrin and Syntaxin 3. Hepatocytes in the MVID patients displayed aberrant canalicular expression of the essential transporters MRP2 and BSEP. The pancreas showed small fragmented islets and a decrease in apical ezrin in pancreatic ducts. Kidney showed normal primary cilia. CONCLUSIONS: These findings indicate that the effects of the P660L mutation in MYO5B in Navajo MVID patients are not limited to the small intestine, but that certain tissues may be able to compensate functionally for alterations in apical trafficking.

Quality indicators for pediatric colonoscopy: results from a multicenter consortium.

BACKGROUND AND AIMS: Currently, there are no quality measures specific to children undergoing GI endoscopy. We aimed to determine the baseline quality of pediatric colonoscopy by using the Pediatric Endoscopy Database System-Clinical Outcomes Research Initiative (PEDS-CORI), a central registry. METHODS: We conducted prospective data collection by using a standard computerized report generator and central registry (PEDS-CORI) to examine key quality indicators from 14 pediatric centers between January 2000 and December 2011. Specific quality indicators, including bowel preparation, ileal intubation rate, documentation of American Society of Anesthesiologists Physical Status Classification System (ASA) class, and procedure time, were compared during the study period. RESULTS: We analyzed 21,807 colonoscopy procedures performed in patients with a mean age of 11.5 ± 4.8 years. Of the 21,807 reports received during the study period, 56% did not include bowel preparation quality, and 12.7% did not include ASA classification. When bowel preparation was reported, the quality was described as excellent, good, or fair in 90.3%. The overall ileal intubation rate was 69.4%, and 15.6% reported cecal intubation only, calculated to be 85% cecum or ileum intubation. Thus, 15% of colonoscopy procedures did not report reaching the cecum or ileum. When excluding the proportion of procedures not intended to reach the ileum (31.5%), the overall ileal intubation rate increased to 84.0%. The rate of ileum examination varied from 85% to 95%, depending on procedure indication. CONCLUSIONS: Colonoscopy reports from our central registry revealed significant variations and inconsistent documentation in pediatric colonoscopy. Our study identifies areas for quality improvement and highlights the need for developing accepted quality measures specific to pediatric endoscopy.

Therapy of caustic ingestion: new treatment considerations.

PURPOSE OF REVIEW: This review will focus on therapeutic considerations and recent advances in treatment of caustic ingestion injuries. RECENT FINDINGS: A retrospective study suggests that it may be safe to advance the endoscope beyond the first circumferential burn to allow for a more complete assessment of extent of injury. A randomized controlled prospective study suggested that a 3-day course of high-dose methylprednisolone might reduce the occurrence of esophageal stricture formation. Balloon dilatation has been shown to be as effective as other bougienage techniques with lower risk of perforations. Recent studies indicate that esophageal dilatation can be safely performed as early as 5-15 days after initial ingestion and may decrease risk for long-term stricture formation. The use of adjunctive treatment, such as topical mitomycin C and esophageal stents, shows promise in reducing the reoccurrence of stricture formation after dilatation. SUMMARY: Caustic ingestion remains a significant problem in children, despite continued efforts to educate the public about ways to avoid this preventable accident. Because there are few good quality therapeutic trials in children, many of the current recommendations regarding treatment are based on expert opinion. Large, prospective, multicenter, controlled treatment trials are needed to identify the best protocols to prevent serious complications.

Rab11a regulates syntaxin 3 localization and microvillus assembly in enterocytes.

Rab11a is a key component of the apical recycling endosome that aids in the trafficking of proteins to the luminal surface in polarized epithelial cells. Utilizing conditional Rab11a-knockout specific to intestinal epithelial cells, and human colonic epithelial CaCo2-BBE cells with stable Rab11a knockdown, we examined the molecular and pathological impact of Rab11a deficiency on the establishment of apical cell polarity and microvillus morphogenesis. We demonstrate that loss of Rab11a induced alterations in enterocyte polarity, shortened microvillar length and affected the formation of microvilli along the lateral membranes. Rab11a deficiency in enterocytes altered the apical localization of syntaxin 3. These data affirm the role of Rab11a in apical membrane trafficking and the maintenance of apical microvilli in enterocytes.

Myosin Vb uncoupling from RAB8A and RAB11A elicits microvillus inclusion disease.

Microvillus inclusion disease (MVID) is a severe form of congenital diarrhea that arises from inactivating mutations in the gene encoding myosin Vb (MYO5B). We have examined the association of mutations in MYO5B and disruption of microvillar assembly and polarity in enterocytes. Stable MYO5B knockdown (MYO5B-KD) in CaCo2-BBE cells elicited loss of microvilli, alterations in junctional claudins, and disruption of apical and basolateral trafficking; however, no microvillus inclusions were observed in MYO5B-KD cells. Expression of WT MYO5B in MYO5B-KD cells restored microvilli; however, expression of MYO5B-P660L, a MVID-associated mutation found within Navajo populations, did not rescue the MYO5B-KD phenotype but induced formation of microvillus inclusions. Microvilli establishment required interaction between RAB8A and MYO5B, while loss of the interaction between RAB11A and MYO5B induced microvillus inclusions. Using surface biotinylation and dual immunofluorescence staining in MYO5B-KD cells expressing mutant forms of MYO5B, we observed that early microvillus inclusions were positive for the sorting marker SNX18 and derived from apical membrane internalization. In patients with MVID, MYO5B-P660L results in global changes in polarity at the villus tips that could account for deficits in apical absorption, loss of microvilli, aberrant junctions, and losses in transcellular ion transport pathways, likely leading to the MVID clinical phenotype of neonatal secretory diarrhea.

Navajo microvillous inclusion disease is due to a mutation in MYO5B.

Microvillous Inclusion Disease (MID) is a rare, autosomal recessive gastrointestinal disease of increased frequency among the Navajos. Previous work has shown a deficiency of RAB8 in one Japanese patient, while homozygous mutations in MYO5B were found in 7 of 10 mostly Middle Eastern families. We have identified a shared homozygous mutation in MYO5B in seven affected Navajos with the expected heterozygosity in five parents. We have developed a simple restriction enzyme based assay that allows for rapid screening for this mutation.

Prevalence of endoscopic findings of erosive esophagitis in children: a population-based study.

PURPOSE: Symptoms of gastroesophageal reflux disease (GERD) occur in 2% to 7% of children. The manifestations of GERD can be limited to symptoms (eg, heartburn, regurgitation) or can be more complicated, such as erosive esophagitis, esophageal strictures, or Barrett esophagus. The prevalence of such GERD complications in children is unknown. The purpose of this study was to determine the prevalence of endoscopic findings of erosive esophagitis in children. PATIENTS AND METHODS: All children ages 0 to 17 years, 11 months who underwent upper endoscopy that was recorded in the Pediatric Endoscopic Database System-Clinical Outcomes Research Initiative between 1999 and 2002 were included. Endoscopic reports that were incomplete or that did not include demographic features, indications for endoscopy, or endoscopic findings were excluded. Erosive esophagitis was defined either descriptively or by the Los Angeles classification. Esophageal biopsy was not evaluated. RESULTS: A total of 7188 children who underwent upper endoscopy fulfilled the inclusion and exclusion criteria. Of those, 888 (12.4%) had erosive esophagitis. The median age of children with erosive esophagitis was 12.7 +/- 4.9 years versus 10.0 +/- 5.1 years in those without erosive esophagitis (P

The prevalence of suspected Barrett's esophagus in children and adolescents: a multicenter endoscopic study.

BACKGROUND: The prevalence of Barrett's esophagus (BE) in young individuals is unclear. OBJECTIVE: To estimate the prevalence of suspected BE in children and adolescent patients undergoing endoscopy. DESIGN: A retrospective cross-sectional study. SETTING: Prospectively collected data in the Pediatric Clinical Outcomes Research Initiative (PEDS-CORI). PATIENTS: We identified patients younger than 20 years of age with suspected BE in the PEDS-CORI between 1999 and 2002; the corresponding histopathologic records were examined. MAIN OUTCOME MEASUREMENTS: We analyzed the distribution of demographic and endoscopic risk factors for BE between cases and non-cases with and without suspected BE in bivariate and multivariable analyses. RESULTS: We identified a total of 6731 patients who underwent upper endoscopy in 12 pediatric facilities. Only 17 patients had suspected BE (prevalence, 2.5 per 1000). Intestinal metaplasia was reported in only 9 of these patients (53%). Patients with suspected BE were older than patients without BE (median 14.7 vs 10.1 years; P = .011). Hiatus hernia was more commonly recorded in patients with suspected BE (11.8% vs 2.2%; P = .008). In a logistic regression model, both older age (odds ratio [OR] 1.13, 95% confidence interval [CI] 1.02-1.35) and hiatus hernia (OR 4.62, 95% CI 1.03-20.66) were independently associated with suspected BE. CONCLUSIONS: Endoscopically suspected BE is rare (<0.25%) in children and adolescents. Older age and the presence of hiatus hernia are possible risk factors for BE in this group. LIMITATIONS: Lack of standardization for identifying and recording endoscopic landmarks.