Genetic background-dependent abnormalities of the enteric nervous system and intestinal function in Kif26a-deficient mice.

The Kif26a protein-coding gene has been identified as a negative regulator of the GDNF-Ret signaling pathway in enteric neurons. The aim of this study was to investigate the influence of genetic background on the phenotype of Kif26a-deficient (KO, -/-) mice. KO mice with both C57BL/6 and BALB/c genetic backgrounds were established. Survival rates and megacolon development were compared between these two strains of KO mice. Functional bowel assessments and enteric neuron histopathology were performed in the deficient mice. KO mice with the BALB/c genetic background survived more than 400 days without evidence of megacolon, while all C57BL/6 KO mice developed megacolon and died within 30 days. Local enteric neuron hyperplasia in the colon and functional bowel abnormalities were observed in BALB/c KO mice. These results indicated that megacolon and enteric neuron hyperplasia in KO mice are influenced by the genetic background. BALB/c KO mice may represent a viable model for functional gastrointestinal diseases such as chronic constipation, facilitating studies on the underlying mechanisms and providing a foundation for the development of treatments.

Mast Cells and Serotonin Synthesis Modulate Chagas Disease in the Colon: Clinical and Experimental Evidence.

BACKGROUND: Trypanosoma cruzi (T. cruzi) infects millions of Latin Americans each year and can induce chagasic megacolon. Little is known about how serotonin (5-HT) modulates this condition. Aim We investigated whether 5-HT synthesis alters T. cruzi infection in the colon. MATERIALS AND METHODS: Forty-eight paraffin-embedded samples from normal colon and chagasic megacolon were histopathologically analyzed (173/2009). Tryptophan hydroxylase 1 (Tph1) knockout (KO) mice and c-KitW-sh mice underwent T. cruzi infection together with their wild-type counterparts. Also, mice underwent different drug treatments (16.1.1064.60.3). RESULTS: In both humans and experimental mouse models, the serotonergic system was activated by T. cruzi infection (p < 0.05). While treating Tph1KO mice with 5-HT did not significantly increase parasitemia in the colon (p > 0.05), rescuing its synthesis promoted trypanosomiasis (p < 0.01). T. cruzi-related 5-HT release (p < 0.05) seemed not only to increase inflammatory signaling, but also to enlarge the pericryptal macrophage and mast cell populations (p < 0.01). Knocking out mast cells reduced trypanosomiasis (p < 0.01), although it did not further alter the neuroendocrine cell number and Tph1 expression (p > 0.05). Further experimentation revealed that pharmacologically inhibiting mast cell activity reduced colonic infection (p < 0.01). A similar finding was achieved when 5-HT synthesis was blocked in c-KitW-sh mice (p > 0.05). However, inhibiting mast cell activity in Tph1KO mice increased colonic trypanosomiasis (p < 0.01). CONCLUSION: We show that mast cells may modulate the T. cruzi-related increase of 5-HT synthesis in the intestinal colon.

Megacystis, megacolon, and malrotation: a new syndromic association?

Chronic intestinal pseudo-obstruction (CIPO) can occur as a consequence of neuropathies including diffuse Intestinal Neuronal Dysplasia (IND), a relatively rare enteric nervous system (ENS) abnormality. Although various authors reported of diffuse IND associated either with intestinal malrotation or megacystis, the co-existence of these three entities in the same patient has never been described before. The aim of this paper is to report for the first time in literature a series of patient with such association, focusing on one who carries a de novo duplication of chromosome 12, suggesting a new syndromic association (megacolon, megacystis, malrotation).

Murine model of Hirschsprung-associated enterocolitis II: Surgical correction of aganglionosis does not eliminate enterocolitis.

BACKGROUND: Hirschsprung disease (HD) results from aganglionosis of the colon, is linked to acute and chronic enterocolitis (known as Hirschsprung-associated enterocolitis) despite successful corrective surgery, and can lead to bacteremia and even death. The genetic and molecular mechanisms underlying these disorders are largely unknown. METHODS: We developed a microsurgical corrective pull-through procedure in mice, and applied that to Ednrb(-/-) mice, which manifest aganglionic megacolon that is very similar to HD. Wild-type littermates (Ednrb(+/+)) also underwent identical surgery. At prespecified time points postoperatively, mice were sacrificed, and histopathologic analyses of intestinal inflammation were performed. Mice of both genotypes were sacrificed after the postoperative recovery period to determine if corrective surgery itself caused inflammation. Stooling patterns were assessed as well to determine if intestinal function normalized after surgery. RESULTS: There was no difference in histopathological enterocolitis scores after recovery from surgery. Stooling patterns in Ednrb(-/-) and Ednrb(+/+) mice were similar postoperatively, suggesting normalization of intestinal function. However, with time, approximately 40% of Ednrb(-/-) mice developed clinical illness consistent with enterocolitis. No control (Ednrb(+/+)) mice developed clinical enterocolitis. Histopathological enterocolitis scores in the 40% of Ednrb(-/-) mice that developed clinical enterocolitis postoperatively were significantly worse than those of healthy postoperative Ednrb(-/-) mice. In contrast, none of the Ednrb(+/+) control mice exhibited postoperative long-term inflammation. CONCLUSIONS: Microsurgical pull-through operation in Ednrb(-/-) mice produces a mouse model that closely resembles key features of Hirschsprung-associated enterocolitis, enabling controlled study of genetic and molecular mechanisms in Ednrb(-/-) mice and other genotypes that produce similar phenotypes.

Ncx (Enx, Hox11L.1) is required for neuronal cell death in enteric ganglia of mice.

BACKGROUND/PURPOSE: Ncx (Enx, Hox11L.1)-deficient (Ncx-/-) mice develop mega-ileo-ceco-colon with a larger number of neuronal cells in the enteric ganglia. We investigated mechanisms related to this abnormality and directed our attention to the effects on gastrointestinal tract functions. METHODS: The number of NADPH diaphorase or cuprolinic blue-positive neuronal cells in the enteric ganglia was examined during growth of the mice. Neuronal cell death of enteric ganglia was assayed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling. Function of the gastrointestinal tract was determined by measuring excretion time of the barium chloride given into the stomach. RESULTS: The number of neuronal cells decreased in control mice older than 2 weeks, and neuronal cell death was evident in the ganglia. However, the number of neuronal cells did not decrease in Ncx-/- mice, and cell death was rare. Excretion time of barium chloride was prolonged in all Ncx-/- mice examined and was improved by the administration of an inhibitor of nitric oxide synthase. CONCLUSIONS: Ncx participates in cell death of enteric neurons. Motor abnormality of the gastrointestinal tract in Ncx-/- mice may be attributed to the large number of neuronal cells.

Foxf1 and Foxf2 control murine gut development by limiting mesenchymal Wnt signaling and promoting extracellular matrix production.

Development of the vertebrate gut is controlled by paracrine crosstalk between the endodermal epithelium and the associated splanchnic mesoderm. In the adult, the same types of signals control epithelial proliferation and survival, which account for the importance of the stroma in colon carcinoma progression. Here, we show that targeting murine Foxf1 and Foxf2, encoding forkhead transcription factors, has pleiotropic effects on intestinal paracrine signaling. Inactivation of both Foxf2 alleles, or one allele each of Foxf1 and Foxf2, cause a range of defects, including megacolon, colorectal muscle hypoplasia and agangliosis. Foxf expression in the splanchnic mesoderm is activated by Indian and sonic hedgehog secreted by the epithelium. In Foxf mutants, mesenchymal expression of Bmp4 is reduced, whereas Wnt5a expression is increased. Activation of the canonical Wnt pathway -- with nuclear localization of beta-catenin in epithelial cells -- is associated with over-proliferation and resistance to apoptosis. Extracellular matrix, particularly collagens, is severely reduced in Foxf mutant intestine, which causes epithelial depolarization and tissue disintegration. Thus, Foxf proteins are mesenchymal factors that control epithelial proliferation and survival, and link hedgehog to Bmp and Wnt signaling.

Evaluation of Hox11L1 in the fmc/fmc rat model of chronic intestinal pseudo-obstruction.

BACKGROUND/PURPOSE: The spontaneous rat mutation, familial megacecum and colon (fmc), is responsible for an autosomal recessive phenotype similar to intestinal pseudo-obstruction observed in Hox11L1-/- mice. We hypothesized that fmc is a mutant allele of the rat Hox11L1 gene and tested this hypothesis by direct sequencing. METHODS: DNA was extracted from fmc/fmc rats and wild-type littermates. All exons, introns, and DNA 5' to the transcriptional start site of rat Hox11L1 were directly sequenced, and data from the mutant and wild-type animals were compared with each other and corresponding genomic data from humans and mice. RESULTS: Alignment of sequences obtained from rat, human, and mouse indicates that putative regulatory elements of the Hox11L1 gene are conserved in rat, mice, and humans. No mutations were identified in the Hox11L1 allele of fmc/fmc rats. CONCLUSIONS: Despite the phenotypic similarities between fmc/fmc rats and Hox11L1-/- mice, fmc does not appear to be a mutant allele of the Hox11L1 gene.

Familial visceral myopathy with pseudo-obstruction, megaduodenum, Barrett's esophagus, and cardiac abnormalities.

This report describes a new subgroup of familial visceral myopathy. Three patients from within this family were admitted to the hospital with pseudo-obstruction. Barium x-ray, abdominal plain film, esophageal manometry, colonoscopy, gastroscopy, and echocardiography were performed in all siblings for diagnostic evaluation. Two of our patients had surgery because of suspicion of acute abdomen. In one of them, full-thickness biopsy, which was performed during laparotomy, revealed findings that were compatible with familial visceral myopathy. Three siblings from this family with visceral myopathy, in which the parents were consanguineous, had megaduodenum, long-segment Barrett's esophagus, and different cardiac abnormalities.

Targeted expression of SV40 large T-antigen to visceral smooth muscle induces proliferation of contractile smooth muscle cells and results in megacolon.

Many pathological conditions result from the proliferation and de-differentiation of smooth muscle cells leading to impaired contractility of the muscle. Here we show that targeted expression of SV40 large T-antigen to visceral smooth muscle cells in vivo results in increased smooth muscle cell proliferation without de-differentiation or decreased contractility. These data suggest that the de-differentiation and proliferation of smooth muscle cells, seen in many pathological states, may be independently regulated. In the T-antigen transgenic mice the increased smooth muscle cell proliferation results in thickening of the distal colon. Consequently the distal colon becomes hyper-contractile and impedes the flow of digesta through the colon resulting in enlargement of the colon oral to the obstruction. These transgenic mice thus represent a novel model of megacolon that results from increased smooth muscle cell proliferation rather than altered neuronal innervation.

A novel pathogenesis of megacolon in Ncx/Hox11L.1 deficient mice.

The Ncx/Hox11L.1 gene, a member of the Hox11 homeobox gene family, is mainly expressed in neural crest-derived tissues. To elucidate the role of Ncx/Hox11L.1, the gene has been inactivated in embryonic stem cells by homologous recombination. The homozygous mutant mice were viable. These mice developed megacolon with enteric ganglia by age 3-5 wk. Histochemical analysis of the ganglia revealed that the enteric neurons hyperinnervated in the narrow segment of megacolon. Some of these neuronal cells degenerated and neuronal cell death occurred in later stages. We propose that Ncx/Hox11L.1 is required for maintenance of proper functions of the enteric nervous system. These mutant mice can be used to elucidate a novel pathogenesis for human neuronal intestinal dysplasia.

Hypertrophy of colonic smooth muscle: structural remodeling, chemical composition, and force output.

The cellular basis of adaptations occurring during the development of megacolon was studied with the lethal spotted mouse model. Age-dependent changes in the length-force characteristics of the colon reach a steady state by 3-4 mo and include an increased relative force development at very short muscle lengths. In megacolon the following occur: 1) structural remodeling expressed as a greater increase in the fraction of maximum force production at short lengths, a shift of optimum length (Lo) to longer lengths, and no change in force per square centimeter; 2) hypertrophy and hyperplasia of both circular and longitudinal muscle; 3) high resting compliance consistent with no disproportionate change in collagen or elastin composition; 4) marked distension so that in situ circumference approximately 1.8 Lo, where active force production is low, and 5) slack length approximately 0.65 Lo, as in normal colon. Biochemical remodeling in megacolon includes disproportionate increases in ATP and phosphocreatine concentration, with 3.5-fold more preformed phosphagen than in normal colon. The myosin concentration is the same in both muscles, but the actin concentration is 1.5-fold greater in megacolon. Most of the cellular changes in megacolon would facilitate high active force output from the muscle at much larger intestinal diameters.

Hypertrophy of colonic smooth muscle: contractile proteins, shortening velocity, and regulation.

Smooth muscle in megacolon was studied in the lethal spotted mouse and its normal sibling. In megacolon, structural remodeling and a very large increase in total protein content are associated with some changes in the contractile protein isoform composition. 1) There is a higher actin concentration in megacolon, primarily caused by a larger proportion of gamma-isoforms. 2) There was no difference in myosin concentration or in SM1/SM2 heavy chains in megacolon and normal muscle contractile proteins. 3) Only LC17a essential light chain is present in both normal and megacolon. 4) The 25- to 50-kDa 5'-insert occurs in 15-20% of the myosin in normal colon, compared with 5- to 10-fold lower amounts in megacolon. In permeabilized muscles there was no significant difference in unloaded shortening velocity (Vo) with maximal thiophosphorylation of the 20-kDa light chains, nor was there significant difference in the force vs. Ca2+ and force vs. myosin light chain phosphorylation relationships. At approximately 60% myosin light chain phosphorylation after Ca2+ activation, Vo of megacolon was approximately two times faster than Vo of normal muscle. These cellular changes largely account for the higher propulsive velocity of the colon in situ. The distribution of myosin and actin isoforms and the lack of a simple relationship between myosin light chain phosphorylation and Vo point to the operation of additional regulatory processes.

Enx (Hox11L1)-deficient mice develop myenteric neuronal hyperplasia and megacolon.

The isolated homeobox gene Enx (Hox11L1) is expressed in enteric neurons innervating distal ileum, and proximal and distal colon. Enx-deficient mice develop megacolon with massive distension of the proximal colon. The number of myenteric ganglia, total neurons per ganglion, and NADPH diaphorase presumptive inhibitory neurons per ganglion are increased in the proximal and distal colon, but decreased in the distal ileum of all Enx-/- mice. Enx-/- mice provide a model for human neuronal intestinal dysplasia (NID), in which myenteric neuronal hyperplasia and megacolon are seen. These results suggest that Enx is required for the proper positional specification and differentiative cell fate of enteric neurons.

Human homology and candidate genes for the Dominant megacolon locus, a mouse model of Hirschsprung disease.

Hirschsprung disease (HSCR) is a congenital disorder of the enteric nervous system characterized by the absence of enteric ganglia. Three genes for HSCR have been identified: the RET proto-oncogene, the gene coding for the endothelin B receptor (EDNRB), and the endothelin 3 gene (EDN3). In mice, natural and in vitro-induced mutations affecting the Ret, Ednrb, and Edn3 genes generate a phenotype similar to human HSCR. Another model of HSCR disease is the Dominant megacolon (Dom), a spontaneous mouse mutation for which the target gene has not yet been identified. The Dom mutation has been mapped to the middle-terminal region of mouse chromosome 15, between D15Mit68 and D15Mit2. Using new or known polymorphisms for conserved human/mouse genes, we established the homology between the Dom locus and human chromosome 22q12-q13. Two genes, Smstr3 and Adsl, not previously mapped in the mouse genome, were located on mouse Chromosome 15. Three genes (Smstr3, Lgals1, and Pdgfb) are possible Dom candidates, as they do not recombine with the Dom mutation in a 252 Dom/+ animal backcross.

Catamnestic investigations in children with malformations of the gastrointestinal tract and the abdominal wall.

Catamnestic investigations were performed in 288 children with malformations of the gastrointestinal tract and the abdominal wall, among them 41 with oesophageal atresia, 41 with stenosis/atresia of the small and large bowel, 78 with anorectal malformations, 75 with Hirschsprung's disease, 28 with omphalocele and 25 with gastroschisis. In atresias/stenoses of the gastrointestinal tract both sexes were equally affected whereas boys were significantly more often affected in both Hirschsprung's disease (p 0.0005) and omphalocele/gastroschisis (p 0.05). The babies were more frequently preterm (p 0.025) than it was the case in controls. Parental age was not higher than the average parental age at time of delivery. Incidence of malformations was significantly higher in the relatives of our patients than in the average population and reached 20% (p 0.0005) in relatives of children with omphalocele/gastroschisis. Teratogenic effects of alcohol, diseases, X-rays and drugs during pregnancy could be suspected in several instances, whereas adverse effects of smoking, previous abortions and cycle disorders before pregnancy could not be established.

[Familial megacolon (author's transl)]. / Familiäres Megakolon.

The genetic aspects of Hirschsprung's disease are discussed and there is a family incidence of 1 to 6%. Boys are more frequently affected. The mode of genetic transmission is not known. Three families with Hirschsprung's disease are reported with special reference to the transmission of Hirschsprung's disease.

Aarskog's syndrome with Hirschsprung's disease, midgut malrotation, and dental anomalies.

A 23-year-old man with Aarskog's syndrome had Hirschspring's disease, midgut malrotation, a renal cyst, a cartilaginous projection of the pinna, geographic tongue, and dental anomalies. The family history, negative for these features, including several malignancies. Any or all of these features could be considered part of Aarskog's syndrome and may represent anomalies of neural crest development.