Canonical Wnt signals combined with suppressed TGFß/BMP pathways promote renewal of the native human colonic epithelium.

BACKGROUND: A defining characteristic of the human intestinal epithelium is that it is the most rapidly renewing tissue in the body. However, the processes underlying tissue renewal and the mechanisms that govern their coordination have proved difficult to study in the human gut. OBJECTIVE: To investigate the regulation of stem cell-driven tissue renewal by canonical Wnt and TGFß/bone morphogenetic protein (BMP) pathways in the native human colonic epithelium. DESIGN: Intact human colonic crypts were isolated from mucosal tissue samples and placed into 3D culture conditions optimised for steady-state tissue renewal. High affinity mRNA in situ hybridisation and immunohistochemistry were complemented by functional genomic and bioimaging techniques. The effects of signalling pathway modulators on the status of intestinal stem cell biology, crypt cell proliferation, migration, differentiation and shedding were determined. RESULTS: Native human colonic crypts exhibited distinct activation profiles for canonical Wnt, TGFß and BMP pathways. A population of intestinal LGR5/OLFM4-positive stem/progenitor cells were interspersed between goblet-like cells within the crypt-base. Exogenous and crypt cell-autonomous canonical Wnt signals supported homeostatic intestinal stem/progenitor cell proliferation and were antagonised by TGFß or BMP pathway activation. Reduced Wnt stimulation impeded crypt cell proliferation, but crypt cell migration and shedding from the crypt surface were unaffected and resulted in diminished crypts. CONCLUSIONS: Steady-state tissue renewal in the native human colonic epithelium is dependent on canonical Wnt signals combined with suppressed TGFß/BMP pathways. Stem/progenitor cell proliferation is uncoupled from crypt cell migration and shedding, and is required to constantly replenish the crypt cell population.

Iron-deficiency anaemia caused by an enterolith-filled jejunal duplication cyst.

Enterogenous duplication cysts are rare, but recognised, developmental anomalies. Duplications in the midgut are the commonest of these rare anomalies with a high proportion of them being found in the jejunum. Enterolith formation within these duplication cysts is uncommon as is heterotopic mucosa giving rise to peptic ulceration. Mode of presentation depends on whether symptoms due to mass effect or gastrointestinal bleeding predominate. We present a case of iron-deficiency anaemia caused by an enterolith-filled jejunal duplication cyst diagnosed at laparotomy and treated by surgical excision and small bowel resection.

Apolipoprotein E gene polymorphism and colorectal cancer: gender-specific modulation of risk and prognosis.

Apolipoprotein E ( ApoE ) gene polymorphism is a major factor in lipid metabolism. It has been suggested that this polymorphism can modulate colorectal tumour risk. We tested this hypothesis for colorectal cancer (CRC). ApoE genotype was determined in 206 patients with CRC and 353 healthy controls from the East Anglia region of the U.K. Compared with individuals possessing the most common epsilon 3/epsilon 3 genotype, those with the epsilon 2/epsilon 3 genotype had an increased risk of colon cancer [odds ratio (OR) = 1.91; 95% confidence interval 1.05-3.45]. However, this association was strongly affected by gender. Separate analysis of male and female subjects revealed a highly significant association in men (OR = 2.71; 95% confidence interval 1.30-5.65), but no association in women (OR = 1.01; 95% confidence interval 0.37-2.77). Likewise, the proportion of male patients with more advanced tumours (Dukes' C&D) was significantly increased among those with the ApoE epsilon 2/epsilon 3 genotype (OR = 4.16; 95% confidence interval 1.36-12.75). No significant effect of the presence of the epsilon 4 allele on CRC risk was found; however, there were no epsilon 4/epsilon 4 homozygotes among patients with proximal colon cancers. The ApoE epsilon 3/epsilon 3 majority genotype appeared to be associated with the lowest risk of CRC. These results suggest that ApoE genotype can influence both CRC risk and prognosis of the existing disease in a gender-dependent manner.

Plasminogen activator inhibitor-1 gene polymorphism and colorectal cancer risk and prognosis.

Plasminogen activator inhibitor-1 (PAI-1) is a factor in urokinase-type plasminogen activator proteolytic system, which is important for tumour invasion and metastasis. Elevated PAI-1 levels in tumours are associated with poor prognosis. An insertion/deletion (4G/5G) promoter polymorphism in the PAI-1 gene affects activity of its product, the 4G/4G genotype being related to higher transcription levels. We assessed the association between the polymorphism and colorectal cancer risk in 206 cancer patients and 355 healthy controls. The results indicated that the PAI-1 gene polymorphism did not affect colorectal cancer risk. Nevertheless, within the case group, the 4G/4G genotype was associated with more advanced tumours (Dukes' C&D), whereas the 5G/5G homozygocity was associated with the Dukes' A&B tumours. The association with the 4G/4G presence was stronger in patients with proximal colon cancers (odds ratio= 6.02, 95% confidence interval, 1.15-31.54). Our results suggest that PAI-1 genotype may be a useful prognostic marker for colorectal cancer, however further specifically designed studies are needed to assess its value in this respect.

The colon-selective spasmolytic otilonium bromide inhibits muscarinic M(3) receptor-coupled calcium signals in isolated human colonic crypts.

1. Otilonium bromide (OB) is a smooth muscle relaxant used in the treatment of irritable bowel syndrome. Otilonium bromide has been shown to interfere with the mobilization of calcium in intestinal smooth muscle, but the effects on other intestinal tissues have not been investigated. We identified the muscarinic receptor subtype coupled to calcium signals in colonic crypt derived from the human colonic epithelium and evaluated the inhibitory effects of OB. 2. Calcium signals were monitored by fluorescence imaging of isolated human colonic crypts and Chinese hamster ovary cells stably expressing the cloned human muscarinic M(3) receptor subtype (CHO-M(3)). Colonic crypt receptor expression was investigated by pharmacological and immunohistochemical techniques. 3. The secretagogue acetylcholine (ACh) stimulated calcium mobilization from intracellular calcium stores at the base of human colonic crypts with an EC(50) of 14 micro M. The muscarinic receptor antagonists 4-DAMP, AF-DX 384, pirenzepine and methroctamine inhibited the ACh-induced calcium signal with the following respective IC(50) (pK(b)) values: 0.78 nM (9.1), 69 nM (7.2), 128 nM (7.1), and 2510 nM (5.8). 4. Immunohistochemical analyses of muscarinic receptor expression demonstrated the presence of M(3) receptor subtype expression at the crypt-base. 5. Otilonium bromide inhibited the generation of ACh-induced calcium signals in a dose dependent manner (IC(50)=880 nM). 6. In CHO-M(3) cells, OB inhibited calcium signals induced by ACh, but not ATP. In addition, OB did not inhibit histamine-induced colonic crypt calcium signals. 7. The present studies have demonstrated that OB inhibited M(3) receptor-coupled calcium signals in human colonic crypts and CHO-M(3) cells, but not those induced by stimulation of other endogenous receptor types. We propose that the M(3) receptor-coupled calcium signalling pathway is directly targeted by OB at the level of the colonic epithelium, suggestive of an anti-secretory action in IBS patients suffering with diarrhoea.