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.

Molecular detection and parasite load of Trypanosoma cruzi in digestive tract tissue of Chagas disease patients affected by megacolon.

Chagas disease, caused by the Trypanosoma cruzi parasite in the Americas affects ∼ 7 million people, 30% with cardiac tissue damage and 10-15% with digestive disorders. In this study, we have developed a protocol to detect the presence of the parasite and estimate its load in resected dysfunctional tissue segments of chronically infected patients with digestive megacolon. We have included samples from 43 individuals, 38/5 with positive/negative serology for Chagas disease and digestive syndromes. Samples of 1.5 to 2.0 cm2 were taken from different points of the dysfunctional digestive tract in specialized centres in Cochabamba, Bolivia. T. cruzi cultures were performed by inoculation with NNN-LIT culture medium, and genomic material was obtained from the samples for multiplex qPCR with TaqMan probes targeting satellite nuclear DNA. Cultures failed to isolate T. cruzi but qPCR reached a sensitivity of 42.1% (16/38) with all three spots and in triplicate. A new quantification methodology using synthetic satellite DNA as quantitation standard revealed parasite loads ranging from 2.2 × 102 to 1.0 × 106 satellite DNA copies/µl. Positive samples from the distal end showed a higher parasite load. The results of the present study strengthen and add further evidence to previous findings in an experimental mouse model of chronic T. cruzi infection, providing a valuable tool to improve scientific knowledge on the relevance of the digestive tract in parasite persistence, and underline the need of a better understanding of host-pathogen interaction in digestive tissues, considering pathophysiology, disease immunology and response to treatment.

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).

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.

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.

Familial chronic megacolon presenting in childhood or adulthood: Seeking the presumed gene association.

OBJECTIVE: We identified a pedigree over five generations with 49 members, some of whom had chronic megacolon presenting in adolescence or adulthood. We aimed to assess the genetic cause of chronic megacolon through clinical and DNA studies. DESIGN: After ethical approval and informed consent, family members provided answers to standard bowel disease questionnaires, radiological or surgical records, and DNA (buccal mucosal scraping). Exome DNA sequencing of colon tissue or blood DNA from seven family members with colon or duodenal dilatation, or no megacolon (n = 1) was carried out. Sanger sequencing was performed in 22 additional family members to further evaluate candidate variants. The study focused on genes of potential relevance to enteric nerve (ENS) maturation and Hirschsprung's disease or megacolon, based on the literature (GFRA1, NKX2-1, KIF26A, TPM3, ACTG2, SCN10A, and C17orf107 [CHRNE]) and other genetic variants that co-segregated with megacolon in the six affected family members. RESULTS: Information was available in all except five members alive at time of study; among 30 members who provided DNA, six had definite megacolon, one megaduodenum, seven significant constipation without bowel dilatation, and 16 normal bowel function by questionnaire. Among genes studied, SEMA3F (g.3:50225360A>G; c1873A>G) was found in 6/6 family members with megacolon. The SEMA3F gene variant was assessed as potentially pathogenic, based on M-CAP in silico prediction. SEMA3F function is associated with genes (KIT and PDGFRB) that impact intestinal pacemaker function. CONCLUSION: Familial chronic megacolon appears to be associated with SEMA3F, which is associated with genes impacting enteric nerve or pacemaker function.

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.

Fine structure mapping and deletion analysis of the murine piebald locus.

piebald (s) is a recessive mutation that affects the development of two cell types of neural crest origin: the melanocytes, responsible for pigment synthesis in the skin, and enteric ganglia, which innervate the lower bowel. As a result, mice carrying piebald mutations exhibit white spotting in the coat and aganglionic megacolon. Previously the gene had been localized to the distal half of mouse chromosome 14. To determine its precise location relative to molecular markers, an intersubspecific backcross was generated. Two anchor loci of chromosome 14, slaty and hypogonadal, in addition to simple sequence length repeat markers, were used to localize s to a 2-cM interval defined by the markers D14Mit38 and D14Mit42. The molecular markers were also used to characterize nine induced s alleles. Three of these mutations exhibited no deletions or rearrangements of the flanking markers, whereas the other six had two or more of these markers deleted. The extent of the deletions was found to be consistent with the severity of the homozygous phenotype. The location of deletion breakpoints in the induced alleles, coupled with the recombination breakpoints in the backcross progeny, provide useful molecular landmarks to define the location of the piebald gene.

Piebald mutation on a C57BL/6J background.

The classic piebald mutation in the endothelin receptor type B (Ednrb) gene was found on rolling Nagoya genetic background (PROD-s/s) mice with white coat spotting. To examine whether genetic background influenced the phenotype in the piebald mutant mice, we generated a congenic strain (B6.PROD-s/s), produced by repeated backcrosses to the C57BL/6J (B6) strain. Although B6.PROD-s/s mice showed white coat spotting, 7% of B6.PROD-s/s mice died between 2 and 5 weeks after birth due to megacolon. The PROD-s/s, s/s and Japanese fancy mouse 1 (JF1) strains, which also have piebald mutations on different genetic backgrounds with B6, showed only pigmentation defects without megacolon. In expression analyses, rectums of B6.PROD-s/s with megacolon mice showed ~5% of the level of Ednrb gene expression versus B6 mice. In histological analyses, aganglionosis was detected in the rectum of megacolon animals. The aganglionic rectum was thought to lead to severe constipation and intestinal blockage, resulting in megacolon. We also observed an abnormal intestinal flora, including a marked increase in Bacteroidaceae and Erysipelotrichaceae and a marked decrease in Lactobacillus and Clostridiales, likely inducing endotoxin production and a failure of the mucosal barrier system, leading ultimately to death. These results indicate that the genetic background plays a key role in the development of enteric ganglion neurons, controlled by the Ednrb gene, and that B6 has modifier gene (s) regarding aganglionosis.

Late infantile Hirschsprung disease-mental retardation syndrome with a 3-bp deletion in ZFHX1B.

A 48-year-old woman with late infantile onset mental retardation developed megacolon. Although the patient had no typical clinical features of Hirschsprung disease-mental retardation syndrome, a new 3-base pair deletion, eliminating an Asn, was identified in the responsible gene ZFHX1B. This suggests that screening for ZFHX1B mutations is warranted even in the absence of typical clinical features of the syndrome.

Neuronal defects in genotyped dominant megacolon (Dom) mouse embryos, a model for Hirschsprung disease.

Dominant megacolon (Dom) is one of four mutations in the mouse which can produce a phenotype similar to Hirschsprung disease in man. Here, we report that it is possible to take advantage of two microsatellite markers to genotype Dom embryos and to study enteric neuronal development in Dom embryos using whole-mount immunohistochemistry. Dom embryos present a variable defect in the ileo-caecal region, as do embryos of other murine models of Hirschsprung disease.

Idiopathic slow transit constipation and megacolon are not associated with neurturin mutations.

Chronic idiopathic slow-transit constipation (ISTC) and idiopathic megacolon (IMC) are early-onset gastrointestinal motility disorders of unknown aetiology. The gene encoding the neurotrophic factor neurturin may be a candidate for these disorders, as neurturin-deficient mice have a similar enteric phenotype. In the present study, we tested this hypothesis. Genomic DNA from 26 cases of chronic idiopathic STC [with a family history of constipation in 15 (58%) and Hirschsprung's disease in two (8%)], and five cases of IMC [two familial (40%)] was screened by direct DNA sequencing using the fluorescent dideoxy terminator method. Results were compared with published sequence data and 24 control DNAs. Our results revealed several previously unreported common sequence polymorphisms, but overall frequencies were comparable between patients and controls. We conclude that mutation of neurturin is not a frequent cause of ISTC or IMC.

Hirschsprung's disease discordant in monozygotic twins: a study of possible environmental factors in the production of colonic aganglionosis.

The occurrence of Hirschsprung's disease, with histologically verified colonic aganglionosis, in only 1 of 2 prematurely born and presumed identical (monozygotic) twins is reported. The occurrence of monozygotic twinning was supported by the observed sharing of a single and common placenta and by ABO and HLA identity of peripheral blood erythrocytes and leukocytes. The affected twin was of a slightly lower birth weight and experienced early respiratory distress, necrotizing enterocolotis, and more prolonged umbilical artery catheterization (no encountered in the unaffected twin). This, to the authors' knowledge, is the first reported occurrence of Hirschsprung's disease discordant in monozygotic twins. The literature relating to genetic and environmental factors in clinical and experimental colonic aganglionosis is reviewed and speculation is presented regarding the occurrence of colonic aganglionosis discordant in monozygotic twins as reported here.

A fetus suggesting an extension of theXK-aprosencephaly spectrum phenotype.

We report a fetus with atelencephaly, bilateral radial aplasia/hypoplasia, ventriculoseptal defect and megacolon, this combination of anomalies being consistent with the diagnosis of XK-aprosencephaly syndrome. The facial dysmorphology of this fetus differs from that previously reported and together with reports on overlapping phenotypes suggests an extension of the XK-aprosencephaly spectrum.

Sequence variability of human cytomegalovirus UL144 open reading frame in low-passage clinical isolates.

OBJECTIVE: To explore the relationship between human cytomegalovirus (HCMV) UL144 sequence variability and clinical disease. METHODS: HCMV UL144 open reading frame (ORF) was amplified by PCR assay in 72 low-passage isolates [65 congenitally infective children and 7 healthy children who were HCMV-DNA positive by quantitative PCR (qPCR)]. All positive PCR products were analyzed by heteroduplex mobility assay and single-stranded conformation polymorphism (HMA-SSCP) and 32 of them were sequenced. RESULTS: Fifty-five patient isolates and five healthy children isolates were HCMV-UL144 positive by PCR. Sequencing and HMA-SSCP analysis showed that significant strain-specific variability was present in the UL144 ORF. Phylogenetic analysis indicated that the nucleotide sequences could be separated into 3 major genotypes. Comparing between UL144 sequences and the corresponding symptoms showed that genotype 2 did not exist in megacolon isolates. And genotype 1 and 3 were the major types among microcephaly and jaundice isolates respectively. CONCLUSIONS: HCMV-UL144 existed in most of low passage isolates and sequences were hypervariable. The UL144 ORF and its predicted product with the high level of sequence variability in different kinds of isolates suggest that UL144 ORF might play a role in HCMV infectivity and subsequent diseases.

[The megacolon in myotonic dystrophy: case report and review of the literature]. / Il megacolon nella distrofia miotonica: case report e review della letteratura.

Myotonic dystrophy (MD) is an autosomal dominant inherit disease, slowly progressive, involving multiple organ systems. Disorders at any level of the gastrointestinal tract are relatively common and manifest as disturbances in motility, such as impaired esophageal transport, delayed gastric emptying, and megacolon. A 51 years-old man was admitted to our surgical department with obstructive symptoms. Diagnostic evaluation showed megacolon and the typical clinical features of the MD, such as weakness, myotonia, frontal baldness and testicular atrophy. Risk of perforation and dehydration led to emergency total colectomy with ileorectal stapled anastomosis. The patient didn't suffer for compliance related to surgical treatment but, after 1 month in intensive care, died of pneumonia and myocardial infarct. The overall frequency of perioperative complications in patients with MD ranges from 8.2 to 42.9%. The risk of perioperative pulmonary complications is particularly high. Thus, we believe that the conservative treatment of motility disorders of the bowel in patients with MD is to be justified and that surgical treatment should be reserved, as last resort, performing a early diagnosis and careful monitoring during perioperative period.

[The problems of breeding spotted rabbits. 3. Variability of the pigmentation grade, ganglionic intestinal wall supply, relationship to pathogenesis--animal breeding and animal welfare aspects]. / Zur Problematik der Scheckenzucht bei Kaninchen. 3. Mitteilung: Variabilität des Pigmentierungsgrades, ganglionäre Darmwandversorgung, Beziehung zur Pathogenese--tierzüchterische und tierschützerische Aspekte.

In a total of 128 animals of the German Giant Spot und the English Spot Breed grade of pigmentation and spotting pattern were investigated by photography and planimetry. A clear cut binary distribution, representing homozygous (KK) or heterozygous spot rabbits (Kk), and a strong bilateral concordance was found as well as a broad variance within both genotypical groups. Thus only a small percentage of animals bred can be compatible with mandatory fancy breed standards. Measurements of intestinal layers and neural components in well pigmented and depigmented individuals revealed an enhancement of these histological structures but a relative hypoganglionosis in distal parts of the gut in animals with predisposition to megacolon (KK, so-called chaplins). It is not yet clear whether this is symptomatic or of primary pathogenetic relevance. There were no pleiotropic deleterious effects of the K-allele in heterozygous rabbits and no correlations between the degree of depigmentation and the severity of symptoms in homozygous spotted ones, thus indicating a mode of inheritance by an incompletely dominant gene K, acting as or being linked to a recessive semilethal when homozygous. A change in breeders' attitude and breeding practices is mandatory because neither the permanent elimination of animals not "fitting" a fancy standard nor the continuous production of defective genotypes is legal.

[The problem of breeding for spots in rabbits. 2. Further results on the variation of characteristics in fattening and breeding animals]. / Zur Problematik der Scheckenzucht bei Kaninchen. 2. Mitteilung: Weitere Ergebnisse zu Merkmalsvarianzen bei Mast- und Zuchttieren.

The mode of inheritance of spotting in both breeds (ES and DRS) is compatible with incompletely dominant resp. intermediate segregation, though a broad variability of spotted areas exists, indicating the influence of minor genes or other ontogenetic factors. Additional findings support the conclusion, that there is a very distinct predisposition of homozygous KK animals to develop megacolon with growing age. This also has consequences for the relative organ weight of the heart and of the adrenals in this genotype. Sporadically encountered accessory adrenal cortical tissue however did not exhibit genotypic preferences.