Showing papers on "Crypt published in 2008"

Loss of intestinal crypt progenitor cells owing to inactivation of both Notch1 and Notch2 is accompanied by derepression of CDK inhibitors p27Kip1 and p57Kip2

Abstract: The crucial role of individual Notch receptors and the mechanism by which they maintain intestinal crypt progenitor cells were assessed by using a series of inducible gut-specific Notch mutant mice. We found that Notch1 and Notch2 receptors function redundantly in the gut, as only simultaneous loss of both receptors results in complete conversion of proliferating crypt progenitors into post-mitotic goblet cells. This conversion correlates with the loss of Hes1 expression and derepression of the cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2. We also found that the promoter of both CDK inhibitor genes is occupied by the Notch effector Hes1 in wild-type crypt progenitor cells. Thus, our results indicate that Notch-mediated Hes1 expression contributes to the maintenance of the proliferative crypt compartment of the small intestine by transcriptionally repressing two CDK inhibitors.

Colonic crypt organization and tumorigenesis

Abstract: Recent advances in our understanding of intestinal crypt biology, including how mutations in stem cells become fixed and expand within the epithelium, has led to new theories on the origins of colonic adenomas and cancers. An appreciation of colonic crypt organization has become essential to any understanding of tumorigenesis in the colon. Intestinal crypts house tissue-specific, multipotential stem cells, which are located in the niche at the base of the intestinal crypt and are capable of regenerating all intestinal cell types. Recent advances in our understanding of crypt biology, including how mutations in stem cells become fixed and expand within the epithelium, has led to new theories on the origins of colonic adenomas and cancers.

Deletion of the WNT target and cancer stem cell marker CD44 in Apc(Min/+) mice attenuates intestinal tumorigenesis.

Abstract: Mutation of the genes encoding the WNT signaling components adenomatous polyposis coli or beta-catenin plays a critical role in the initiation of colorectal cancer. These mutations cause constitutively active beta-catenin/TCF-mediated transcription, driving the transformation of intestinal crypts to colorectal cancer precursor lesions, called dysplastic aberrant crypt foci. CD44 is a prominent WNT signaling target in the intestine and is selectively expressed on the renewing epithelial cells lining the crypts. The expression of CD44 is dramatically increased in aberrant crypt foci in both humans and tumor-susceptible Apc(Min/+) mice, suggesting a role for CD44 in intestinal tumorigenesis. To study this role, we crossed C57BL/6J-Cd44(-/-) mice with C57BL/6J-Apc(Min/+) mice. Compared with C57BL/6J-Cd44(+/+)/Apc(Min/+) mice, C57BL/6J-Cd44(-/-)/Apc(Min/+) mice showed an almost 50% reduction in the number of intestinal adenomas. This reduction was primarily caused by a decrease in the formation of aberrant crypts, implying the involvement of CD44 in tumor initiation. The absence of CD44 in the normal (nonneoplastic) crypts of Cd44(-/-)/Apc(Min/+) mice did not alter the proliferative capacity and size of the intestinal stem cell and transit-amplifying compartments. However, compared with Cd44(+/+)/Apc(Min/+) mice, Cd44(-/-)/Apc(Min/+) showed an increase in the number of apoptotic epithelial cells at the base of the crypt which correlated with an increased expression of the proapoptotic genes Bok and Dr6. Our results show an important role for CD44 in intestinal tumorigenesis and suggest that CD44 does not affect proliferation but is involved in the control of the balance between survival and apoptosis in the intestinal crypt.

How dysregulated colonic crypt dynamics cause stem cell overpopulation and initiate colon cancer.

Abstract: Based on investigation of the earliest colonic tissue alteration in familial adenomatous polyposis (FAP) patients, we present the hypothesis that initiation of colorectal cancer by adenomatous polyposis coli (APC) mutation is mediated by dysregulation of two cellular mechanisms. One involves differentiation, which normally decreases the proportion (proliferative fraction) of colonic crypt cells that can proliferate; the other is a cell cycle mechanism that simultaneously increases the probability that proliferative cells are in S phase. In normal crypts, stem cells (SC) at the crypt bottom generate rapidly proliferating cells, which undergo differentiation while migrating up the crypt. Our modeling of normal crypts suggests that these transitions are mediated by mechanisms that regulate proliferative fraction and S-phase probability. In FAP crypts, the population of rapidly proliferating cells is shifted upwards, as indicated by the labeling index (LI; i.e., crypt distribution of cells in S phase). Our analysis of FAP indicates that these transitions are delayed because the proliferative fraction and S-phase probability change more slowly as a function of crypt level. This leads to expansion of the proliferative cell population, including a subpopulation that has a low frequency of S-phase cells. We previously reported that crypt SC overpopulation explains the LI shift. Here, we determine that SCs (or cells having high stemness) are proliferative cells with a low probability of being in S phase. Thus, dysregulation of mechanisms that control proliferative fraction and S-phase probability explains how APC mutations induce SC overpopulation at the crypt bottom, shift the rapidly proliferating cell population upwards, and initiate colon tumorigenesis.

Glucagon-Like Peptide-2 Activates β-Catenin Signaling in the Mouse Intestinal Crypt: Role of Insulin-Like Growth Factor-I

Abstract: Chronic administration of glucagon-like peptide-2 (GLP-2) induces intestinal growth and crypt cell proliferation through an indirect mechanism requiring IGF-I. However, the intracellular pathways through which IGF-I mediates GLP-2-induced epithelial tropic signaling remain undefined. Because beta-catenin and Akt are important regulators of crypt cell proliferation, we hypothesized that GLP-2 activates these signaling pathways through an IGF-I-dependent mechanism. In this study, fasted mice were administered Gly(2)-GLP-2 or LR(3)-IGF-I (positive control) for 0.5-4 h. Nuclear translocation of beta-catenin in non-Paneth crypt cells was assessed by immunohistochemistry and expression of its downstream proliferative markers, c-myc and Sox9, by quantitative RT-PCR. Akt phosphorylation and activation of its targets, glycogen synthase kinase-3beta and caspase-3, were determined by Western blot. IGF-I receptor (IGF-IR) and IGF-I signaling were blocked by preadministration of NVP-AEW541 and through the use of IGF-I knockout mice, respectively. We found that GLP-2 increased beta-catenin nuclear translocation in non-Paneth crypt cells by 72 +/- 17% (P < 0.05) and increased mucosal c-myc and Sox9 mRNA expression by 90 +/- 20 and 376 +/- 170%, respectively (P < 0.05-0.01), with similar results observed with IGF-I. This effect of GLP-2 was prevented by blocking the IGF-IR as well as ablation of IGF-I signaling. GLP-2 also produced a time- and dose-dependent activation of Akt in the intestinal mucosa (P < 0.01), most notably in the epithelium. This action was reduced by IGF-IR inhibition but not IGF-I knockout. We concluded that acute administration of GLP-2 activates beta-catenin and proliferative signaling in non-Paneth murine intestinal crypt cells as well as Akt signaling in the mucosa. However, IGF-I is required only for the GLP-2-induced alterations in beta-catenin.

Ectopic Expression of P-Cadherin Correlates with Promoter Hypomethylation Early in Colorectal Carcinogenesis and Enhanced Intestinal Crypt Fission In vivo

Abstract: P-cadherin is normally expressed in the basal layer of squamous epithelia and absent from the healthy intestine and colon. We have previously shown it to be expressed in all inflamed, hyperplastic, and dysplastic intestinal and colonic mucosa. This study aimed to better understand the mechanisms controlling the expression of P-cadherin and the biological effects of its ectopic presence in the intestine and colon. We investigated the CpG methylation status of the P-cadherin (CDH3) promoter and P-cadherin mRNA and protein expression in cases of familial and sporadic colorectal cancer (CRC). The CDH3 promoter was hypomethylated in colonic aberrant crypt foci, in CRC, and, occasionally, in the normal epithelium adjacent to cancer, demonstrating a potential "field effect" of cancerization. The hypomethylation was also associated with induction of P-cadherin expression in the neoplastic colon (P < 0.0001). We then created transgenic mice that overexpressed P-cadherin specifically in the intestinal and colonic epithelium under the liver fatty acid binding protein promoter. Forced ectopic expression of P-cadherin accompanied by indomethacin-induced inflammation resulted in a 3-fold higher crypt fission rate within the small and large intestines in the homozygous mice compared with the wild-type animals (P < 0.02). We conclude that epigenetic demethylation of the P-cadherin promoter in the human intestine permits its ectopic expression very early in the colorectal adenoma-carcinoma sequence and persists during invasive cancer. Induced P-cadherin expression, especially in mucosal damage, leads to an increased rate of crypt fission, a common feature of clonal expansion in gastrointestinal dysplasia.

Protective effect of phlorotannin components phloroglucinol and eckol on radiation-induced intestinal injury in mice.

Abstract: Components of phlorotannin, which were extracted from Ecklonia species, have been reported to have in vitro radioprotective and antioxidative effects. The radioprotective effects of two of the components of phlorotannin, phloroglucinol and eckol, in intestinal stem cells were examined by evaluating their effects on jejunal crypt survival and apoptosis in gamma-irradiated mice. Pretreating the mice (i.p. 20 mg/kg of body weight at 12 and 36 h before irradiation) prior to irradiation with either phloroglucinol or eckol significantly improved the survival of the jejunal crypt (p < 0.001 and p < 0.01 vs irradiation controls at 3.5 days after 8 Gy irradiation, respectively). The administration of phloroglucinol and eckol prior to irradiation protected the intestinal crypts from radiation-induced apoptosis (p < 0.05 vs irradiation controls at 12 h after 1 Gy irradiation). Although the mechanism for this inhibitory effect remains unknown, these results showed that phloroglucinol and eckol might be useful radioprotectors that can defend intestinal stem cells against the oxidative damage caused by gamma-irradiation.

Insulin receptor substrate-1 deficiency promotes apoptosis in the putative intestinal crypt stem cell region, limits Apcmin/+ tumors, and regulates Sox9.

Abstract: Reduced apoptosis of crypt stem/progenitor cells and elevated insulin and IGFs are linked to colon cancer risk. Insulin receptor substrate-1 (IRS-1) mediates the actions of insulin, IGF-I, and IGF-II, but the role of endogenous IRS-1 in crypt apoptosis and cancer is undefined. Using IRS-1−/−, IRS-1+/−, and IRS-1+/+ mice, we tested the hypothesis that reduced IRS-1 expression increases apoptosis of intestinal crypt cells and protects against Apcmin/+ (Min)/β-catenin-driven intestinal tumors. Expression of Sox9, a transcriptional target of Tcf/β-catenin and putative biomarker of crypt stem cells, was assessed in intestine of different IRS-1 genotypes and cell lines. Irradiation-induced apoptosis was significantly increased in the crypts and crypt stem cell region of IRS-1-deficient mice. Tumor load was significantly reduced by 31.2 ± 14.6% in IRS-1+/−/Min and by 64.1 ± 7.6% in IRS-1−/−/Min mice, with more prominent reductions in tumor number than size. Compared with IRS-1+/+/Min, IRS-1−/−/Min mice had fewer Sox9-positive cells in intestinal crypts and reduced Sox9 mRNA in intestine. IRS-1 overexpression increased Sox9 expression in an intestinal epithelial cell line. We conclude that even small reductions in endogenous IRS-1 increase apoptosis of crypt stem or progenitor cells, protect against β-catenin-driven intestinal tumors, and reduce Sox9, a Tcf/β-catenin target and putative stem/progenitor cell biomarker.

The elusive crypt olfactory receptor neuron: evidence for its stimulation by amino acids and cAMP pathway agonists.

Abstract: Crypt olfactory receptor neurons (ORNs) are a third type of chemosensory neuron along with ciliated and microvillous ORNs in the olfactory epithelium of fishes, but their functional role is still unknown. To investigate their odorant response properties and possible transduction pathways, we recorded crypt ORN activity with calcium imaging and the patch clamp technique in its cell-attached mode in combination with odorant and agonist stimulation. Bile salts and putative fish pheromones did not elicit responses with either method, but the cells frequently responded to amino acids, with excitation and intracellular calcium signals. 8Br-cAMP and IBMX plus forskolin stimulated over 40% of crypt ORNs and triggered calcium signals in a similar percentage. Furthermore, crypt ORNs were immunoreactive to an antiserum against adenylate cyclase III. Together, these data suggest the presence of a cAMP transduction pathway, which might transduce odorants such as amino acids.

Dietary heme injures surface epithelium resulting in hyperproliferation, inhibition of apoptosis and crypt hyperplasia in rat colon

Abstract: Epidemiological and animal model studies suggest that a high intake of heme, present in red meat, is associated with an increased risk of colon cancer. The aim of this study was to elucidate the effects of dietary heme on colonic cell homeostasis in rats. Rats were fed a purified, humanized, control diet or a similar diet supplemented with 0.5 mmol heme/kg for 14 days. Fecal water cytolytic activity was determined with a bioassay, and colon epithelial cell proliferation was evaluated with (3)H-thymidine or 5-bromo-2'-deoxyuridine incorporation into DNA or by Ki-67 immunohistochemistry. Exfoliation of colonocytes was measured as the amount of rat DNA in feces, and caspase-3 expression and activity were measured to study colonic mucosal apoptosis. Dietary heme induced a >10-fold increased cytolytic activity of the fecal water and a 100-fold lower excretion of host DNA. Colons of heme-fed rats showed injured surface epithelium and an approximately 25% increase in crypt depth. Finally, dietary heme doubled colonocyte proliferation, shown by all three markers, but inhibited colonic mucosal apoptosis. In conclusion, our results demonstrate that dietary heme injures colonic surface epithelium, which is overcompensated by inhibition of apoptosis and hyperproliferation of cells in the crypts, resulting in crypt hyperplasia. This disturbed epithelial cell homeostasis might explain why a high intake of dietary heme is associated with an increased risk of colon cancer.

In vivo gene expression profiling of human intestinal epithelial cells: analysis by laser microdissection of formalin fixed tissues

Abstract: The small intestinal epithelium mediates vital functions of nutrient absorption and host defense. The spatial organization of the epithelial cells along the crypt-villus axis segregates them into regions of specialized function. However, the differences in transcriptional programming and the molecular machinery that governs the migration, adhesion, and differentiation of intestinal epithelial cell lineages in humans remain under-explored. To increase our understanding of these mechanisms, we have evaluated gene expression patterns of ileal epithelial cells isolated by laser capture microdissection from either the villus epithelial or crypt cell regions of healthy human small intestinal mucosa. Expression profiles in villus and crypt epithelium were determined by DNA microarray, quantitative real-time PCR, and immunohistochemistry based methods. The expression levels of selected epithelial biomarkers were also compared between gastrointestinal tissues. Previously established biomarkers as well as a novel and distinct set of genes believed to be linked to epithelial cell motility, adhesion, and differentiation were found to be enriched in each of the two corresponding cell populations (GEO accession: GSE10629). Additionally, high baseline expression levels of innate antimicrobials, alpha defensin 5 (HD5) and regenerating islet-derived 3 alpha (Reg3A), were detected exclusively within the small bowel crypt, most notably in the ileum in comparison to other sites along the gastrointestinal tract. The elucidation of differential gene expression patterns between crypt and villus epithelial cell lineages in human ileal tissue provides novel insights into the molecular machinery that mediates their functions and spatial organization. Moreover, our findings establish an important framework of knowledge for future investigations of human gastrointestinal diseases.

Crypt fission peaks early during infancy and crypt hyperplasia broadly peaks during infancy and childhood in the small intestine of humans.

Abstract: BACKGROUND Postnatal growth of the small intestine occurs by crypt hyperplasia and by the less recognised mechanism of crypt fission. How the small intestine grows is largely extrapolated from animals and is poorly described in humans. AIM To investigate crypt fission and crypt hyperplasia as mechanisms of intestinal growth in humans. PATIENTS AND METHODS Proximal intestinal samples were taken from 3 neonates at surgical anastomosis, and duodenal biopsies were taken at endoscopy from 16 infants (mean age 0.7, range 0.3-1.7 years), 14 children (mean age 7.9, range 2.4-16.2 years), and 39 adults. Morphometric measures of villous area, crypt length (measure of crypt hyperplasia), and percentage of bifid crypts (measure of crypt fission) were assessed by a microdissection technique. RESULTS Mean crypt fission rates in neonates, infants, children, and adults were 7.8%, 15%, 4.9%, and 1.7%, respectively. In particular, crypt fission peaked at 18% in 5 infants from 6 to 12 months of age. Mean crypt length was 123 microm in neonates, 287 microm in infants, 277 microm in children, and 209 microm in adults. Thus, crypt hyperplasia had a broad peak during infancy and childhood. CONCLUSIONS We conclude that crypt fission was present predominantly during infancy, and crypt hyperplasia occurred during both infancy and childhood.

Activation of NF-κB is required for mediating proliferative and antiapoptotic effects of progastrin on proximal colonic crypts of mice, in vivo

Abstract: Mice overexpressing progastrin (PG) in intestinal mucosa (fatty acid-binding protein (Fabp)-PG mice) are at an increased risk of proximal colon carcinogenesis in response to azoxymethane. Here, we report a significant increase in the length of proximal colonic crypts in Fabp-PG mice, associated with potent antiapoptotic effects of PG, which likely contributed to the previously reported increase in colon carcinogenesis in Fabp-PG mice. Phosphorylation of kinase of IkappaBalpha (IKKalpha/beta), inhibitor of kappaB (IkappaB)alpha and p65NF-kappaB was significantly elevated in proximal colonic crypts of Fabp-PG versus wild-type mice, which was associated with degradation of IkappaBalpha and nuclear translocation/activation of p65. Surprisingly, distal colonic crypt cells were not as responsive to elevated levels of PG in Fabp-PG mice. Annexin II, recently described as a high-affinity receptor for PG, strongly co-localized with PG intracellularly and on basolateral membranes of proximal crypt cells, providing evidence that annexin-II binds PG in situ in colonic crypt cells. Proliferative and antiapoptotic effects of PG on proximal crypts of Fabp-PG mice were attenuated to wild-type levels, on treatment with NEMO peptide (an inhibitor of nuclear factor-kappaB (NF-kappaB) activation), demonstrating for the first time a critical role of NF-kappaB in mediating hyperproliferative affects of PG on colonic crypts of Fabp-PG mice, in vivo. Thus, downregulation of NF-kappaB may significantly reduce the increased risk of colon carcinogenesis in response to PG.

Epithelial and mesenchymal cells in the bovine colonic mucosa differ in their responsiveness to Escherichia coli Shiga toxin 1.

Abstract: Bovine colonic crypt cells express CD77 molecules that potentially act as receptors for Shiga toxins (Stx). The implication of this finding for the intestinal colonization of cattle by human pathogenic Stx-producing Escherichia coli (STEC) remains undefined. We used flow cytometric and real-time PCR analyses of primary cultures of colonic crypt cells to evaluate cell viability, CD77 expression, and gene transcription in the presence and absence of purified Stx1. A subset of cultured epithelial cells had Stx receptors which were located mainly intracellularly, with a perinuclear distribution, and were resistant to Stx1-induced apoptosis and Stx1 effects on chemokine expression patterns. In contrast, a population of vimentin-positive cells, i.e., mesenchymal/nonepithelial cells that had high numbers of Stx receptors on their surface, was depleted from the cultures by Stx1. In situ, CD77+ cells were located in the lamina propria of the bovine colon by using immunofluorescence staining. A newly established vimentin-positive crypt cell line with high CD77 expression resisted the cytolethal effect of Stx1 but responded to Stx1 with a significant increase in interleukin-8 (IL-8), GRO-α, MCP-1, and RANTES mRNA. Combined stimulation with lipopolysaccharide and Stx1 increased IL-10 mRNA. Our results show that bovine colonic crypt cells of epithelial origin are resistant to both the cytotoxic and modulatory effects of Stx1. In contrast, some mucosal mesenchymal cells, preliminarily characterized as mucosal macrophages, are Stx1-responsive cells that may participate in the interaction of STEC with the bovine intestinal mucosa.

Regulation of intestinal goblet cells in situ, in mucosal explants and in the isolated epithelium.

Abstract: Cholinergic secretagogues were previously shown to accelerate mucin secretion from intestinal goblet cells of adult rats and rabbits, both in vitro and in mucosal explants. This rapid secretory response occurs only in crypt cells; surface goblet cells are not affected. Rapid secretion involves the sequential fusion of secretory granule membranes with the plasma membrane and with each other, but does not require granule movement. In unstimulated cells, slow transport of secretory granules towards the luminal cell surface depends on functional microtubules. Goblet cells appear in the rat fetal intestine three to four days before birth but they are insensitive to cholinergic agents in the fetus and neonate. The secretory response of crypt goblet cells to carbachol, both in vivo and in mucosal slices in vitro, is established throughout the intestines only after weaning (20-25 days after birth). To determine whether acetylcholine from nerve endings in the intact mucosa may mediate a mucus secretory response in the absence of exogenous secretagogues, mucosal sheets were mounted in modified Ussing chambers and goblet cell secretion was assessed after electrical field stimulation. Electrical field stimulation elicited mucus secretion from crypt (but not surface) goblet cells. Secretion was inhibited by prior treatment of the mucosa with 500 nM-tetrodotoxin or 100 microM-atropine, but not by 10 microM-atropine. Thus, endogenous nerves may regulate mucus secretion from crypt goblet cells in the intact mucosa. When intact sheets of epithelium were isolated from adult rat ileum and colon, then maintained in vitro and exposed to 20 microM-carbachol, crypt goblet cells released mucin in response to the secretagogue but goblet cells in in portions of the epithelium derived from villi or mucosal surfaces were unresponsive. This suggests that crypt epithelial cells respond directly to cholinergic agents and that they lose this sensitivity as they migrate out of the crypts.

Resistant carbohydrates stimulate cell proliferation and crypt fission in wild-type mice and in the Apc(Min/+) mouse model of intestinal cancer, association with enhanced polyp development.

Abstract: Fermentation of carbohydrates in the colon can stimulate cell proliferation and could thus be a cancer risk. The effects of resistant carbohydrates, i.e. those not digested and absorbed in the small intestine, on cell proliferation, crypt fission and polyp development were investigated in wild-type and adenomatous polyposis coli multiple intestinal neoplasia (Apc(Min/+)) mice. Fifteen 4-week-old female wild-type and fifteen Apc(Min/+) mice were used for each group and fed a chow diet, a semi-synthetic diet or the semi-synthetic supplemented with wheat bran or an apple pomace preparation, both high in resistant carbohydrates, for 8 weeks. Tissue from all mice was used to measure cell proliferation and crypt fission and tissue from the Apc(Min/+) mice was scored for polyp number and tumour burden. There were slight reductions in intestinal mass in the mice fed the semi-synthetic diets and this was increased by the inclusion of resistant carbohydrates. The Apc(Min/+) mice had elevated cell proliferation and crypt fission in the distal small intestine and colon and these were increased by the resistant carbohydrates. Bran or apple pomace significantly increased polyp number in the proximal third of the small intestine. Apple pulp more than doubled polyp number throughout the small bowel (99.2 (SEM 11.1) v. 40.0 (SEM 8.2), P<0.004). Bran and apple pomace increased polyp diameter and hence burden in the colon by 243 and 150 %, respectively (P<0.05). In conclusion, both types of resistant carbohydrates increased polyp number and tumour burden and this was associated with elevated epithelial cell proliferation and crypt fission.

FGFR3 contributes to intestinal crypt cell growth arrest.

Abstract: Fibroblast growth factors (FGFs) are important regulators of the dynamic development and turnover of tissues. Among FGF receptors, FGFR3 expression is confined in the intestinal crypts. We examined FGFR3-deficient mice and saw increased intestinal crypt depth but no change in villae length or in the distribution of differentiated intestinal cells, suggesting that the impact of lack of FGFR3 was limited to the progenitor cell compartment. Accordingly, enhancement of intestinal crypt proliferation was observed in FGFR3 mutant mice and interestingly, upon anti-FGFR3 antibody administration in wild type mice. Moreover, injection of FGF18, a ligand of FGFR3, in wild type mice resulted in decreased cell proliferation within the intestinal crypts. In addition, we found that ERK level of activation was increased in FGFR3-deficient intestinal epithelium. In vitro studies showed that ERK, AKT and activation was regulated by FGFs and that ERK level of activation was inversely correlated to FGFR3 level of expression in the intestinal crypt cells. Furthermore, effects of FGF18 on ERK and AKT activation paralleled FGFR3 effects on these intracellular targets. Our data indicate that FGF18 and FGFR3 are involved, possibly as partners, in the control of intestinal precursor cell proliferation.

Intrinsic denervation of the colon is associated with a decrease of some colonic preneoplastic markers in rats treated with a chemical carcinogen

Abstract: Denervation of the colon is protective against the colon cancer; however, the mechanisms involved are unknown. We tested the hypothesis that the denervated colonic mucosa could be less responsive to the action of the chemical carcinogen dimethylhydrazine (DMH). Three groups of 32 male Wistar rats were treated as follows: group 1 (G1) had the colon denervated with 0.3 mL 1.5 mM benzyldimethyltetradecylammonium (benzalkonium chloride, BAC); G2 received a single ip injection of 125 mg/kg DMH; G3 was treated with BAC + the same dose and route of DMH. A control group (Sham, N = 32) did not receive any treatment. Each group was subdivided into four groups according to the sacrifice time (1, 2, 6, and 12 weeks after DMH). Crypt fission index, ss-catenin accumulated crypts, aberrant crypt foci, and cell proliferation were evaluated and analyzed by ANOVA and the Student t-test. G3 animals presented a small number of aberrant crypt foci and low crypt fission index compared to G2 animals after 2 and 12 weeks, respectively. From the second week on, the index of ss-catenin crypt in G3 animals increased slower than in G2 animals. From the 12th week on, G2 animals presented a significant increase in cell proliferation when compared to the other groups. Colonic denervation plays an anticarcinogenic role from early stages of colon cancer development. This finding can be of importance for the study of the role of the enteric nervous system in the carcinogenic process.

Characterization of ryanodine receptors in rat colonic epithelium

Abstract: Aim: Functional evidence suggests the presence of two types of intracellular Ca2+ channels responsible for the release of Ca2+ from Ca2+-stores, i.e. inositol-1,4,5-trisphosphate (IP3R) and ryanodine receptors (RyR), in rat colonic epithelium. Generally, three ryanodine receptor isoforms (RyR1–RyR3) are known; however, the type of RyR at this epithelium is unknown and was the focus of the present study. Methods: RyRs were characterized by molecular biological and immunohistochemical methods in the rat colon. Results: A transcript of RyR1 was found in mRNA from colonic crypts. In contrast, RyR2 and RyR3 were found in their corresponding reference tissues, but not in the cDNA from colonic crypts suggesting a predominant expression of the RyR1 isoform in this epithelium. In order to characterize the subcellular localization of RyR1, immunohistochemical experiments were performed. They showed that RyR1 is present in the lamina epithelialis mucosae and smooth muscle cells and is distributed equally along the whole crypt axis with no difference between surface and crypt cells. A double staining with IP3R3, the dominant cytoplasmic isoform of IP3Rs in this epithelium, revealed that there is only little colocalization of the two receptor subtypes within the epithelial cells. Furthermore, the epithelium is equipped with the enzyme CD38 responsible for the production of cyclic adenosine diphosphate ribose, the physiological agonist of RyR. RyRs are known to be activated by changes in the redox state. The oxidant, monochloramine evoked a ruthenium red-sensitive Ca2+ release all over the crypt axis. This release was unaffected by prior stimulation of IP3 receptors with ATP (and vice versa). Conclusion: The present data suggest a functional separation of IP3- and ryanodine receptor-carrying Ca2+ stores in the colonic epithelium.

Crypt region localization of intestinal stem cells in adults.

Abstract: The intestinal epithelial lining plays a central role in the digestion and absorption of nutrients, but exists in a harsh luminal environment that necessitates continual renewal. This renewal process involves epithelial cell proliferation in the crypt base and later cell migration from the crypt base to the luminal surface. This process is dependent on multi-potent progenitor cells, or stem cells, located in each crypt. There are about 4 to 6 stem cells per crypt, and these stem cells are believed to generate distinct end-differentiated epithelial cell types, including absorptive cells, goblet cells, enteroendocrine cells and Paneth cells, while also maintaining their own progenitor cell state. Earlier studies suggested that intestinal stem cells were located either in the crypt base interspersed between the Paneth cells [i.e. crypt base columnar (CBC) cell model] or at an average position of 4 cells from the crypt base [i.e. label-retaining cells (LRC +4) model]. Recent studies have employed biomarkers in the in vivo mammalian state to more precisely evaluate the location of these progenitor cells in the intestinal crypt. Most notable of these novel markers are Lgr5, a gene that encodes a G-protein-coupled receptor with expression restricted to CBC cells, and Bmi 1, which encodes a chromatin remodeling protein expressed by LRC. These studies raise the possibility that there may be separate stem cell lines or different states of stem cell activation involved in the renewal of normal mammalian intestinal tract.

β-catenin is strongly elevated in rat colonic epithelium following short-term intermittent treatment with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and a high-fat diet

Abstract: Colon tumors expressing high levels of β-catenin and c-myc have been reported in male F344 rats given three short cycles of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) alternating with a high-fat (HF) diet. Using the same experimental protocol, rats were euthanized 24 h after the last dose of PhIP so as to examine early changes in colonic crypt homeostasis and β-catenin expression, before the onset of frank tumors. PhIP/HF dosing caused a significant increase in the bromodeoxyuridine labeling index throughout the entire colon, and within the colonic crypt column cleaved caspase-3 was elevated in the basal and central zones, but reduced in the luminal region. In vehicle/HF controls, β-catenin was immunolocalized primarily at the border between cells at the top of the crypt, whereas in rats given PhIP/HF diet there was strong cytoplasmic staining, which appeared as a gradient of increased β-catenin extending from the base of the crypt column to the luminal region. Quantitative real-time PCR and immunoblot analyses confirmed that β-catenin and c-myc were increased significantly in the colonic mucosa of rats given PhIP/HF diet. Collectively, these findings suggest that PhIP/HF cycling alters β-catenin and c-myc expression in the colonic mucosa, resulting in expansion of the proliferative zone and redistribution of apoptotic cells from the lumen to the central and basal regions of the colonic crypt. Thus, during the early stages of colon carcinogenesis, alternating exposure to heterocyclic amines and a high-fat diet might facilitate molecular changes resulting in dysregulated β-catenin and c-myc expression. (Cancer Sci 2008; 99: 1754–1759)

Evaluation on Recent Committed Crypt Analysis Hash Function

Abstract: This paper describes the study of cryptographic hash functions, one of the most important classes of primitives used in recent techniques in cryptography. The main aim is the development of recent crypt analysis hash function. We present different approaches to defining security properties more formally and present basic attack on hash function. We recall Merkle-Damgard security properties of iterated hash function. The Main aim of this paper is the development of recent techniques applicable to crypt Analysis hash function, mainly from SHA family. Recent proposed attacks an MD5 & SHA motivate a new hash function design. It is designed not only to have higher security but also to be faster than SHA-256. The performance of the new hash function is at least 30% better than that of SHA-256 in software. And it is secure against any known cryptographic attacks on hash functions. Keywords—Crypt Analysis, cryptographic.

Crypt Dysplasia: Fact or Fiction?

Abstract: The recognition of dysplasia arising in Barrett esophagus can be an exceedingly difficult task. It is well known that there is a significant degree of interobserver variability, particularly at the low end of the Barrett esophagus-dysplasia spectrum (negative for dysplasia vs. indefinite for dysplasia vs. low-grade dysplasia). As stated by Lomo and colleagues, ‘‘It is commonly believed among most gastrointestinal pathologists that, regardless of the grade, true dysplasia is characterized by a distinct lack of surface maturation,’’ a concept strongly advocated by Reid and colleagues in their seminal article published in Human Pathology in 1988. In a fascinating and somewhat controversial study by Lomo and colleagues, this concept has been challenged, as the authors describe a process of crypt dysplasia with surface maturation. These authors studied the clinical, pathologic, immunohistochemical, and molecular characteristics of ‘‘basal crypt dysplasia-like atypia’’ (BCDA) with surface maturation in surveillance biopsies from patients with Barrett esophagus. Of 206 consecutive Barrett esophagus patients evaluated prospectively for BCDA over a period of slightly more than 2 years, 15 patients (7.3%) were found to have BCDA. Interestingly, 13 of these 15 patients (87%) had dysplasia or adenocarcinoma detected in biopsies either before or concurrent to the biopsy that contained BCDA. This is in striking contrast to the control group, in which only 59% had neoplasia detected during this same time period. When compared with adjacent nondysplastic Barrett mucosa, BCDA foci showed a significantly higher prevalence rate of p53 immunoreactivity as well as a significantly elevated total crypt and basal crypt MIB-1 proliferation rate. In fact, the MIB-1 proliferation rate in the basal portion of the BCDA crypts was similar to that detected in conventional low-grade and high-grade dysplasia. In addition to the aforementioned immunohistochemical features, patients with BCDA also showed a significantly increased rate of 17p (TP53) loss of heterozygosity and aneuploidy compared with control patients without BCDA. The authors concluded that BCDA with surface maturation in mucosal biopsies from Barrett esophagus patients is an uncommon but significant finding with proliferative and molecular abnormalities similar to those seen in conventional dysplasia and/or adenocarcinoma. These findings are quite provocative and obviously suggest that surface maturation is not a requirement for the histologic recognition of dysplasia in Barrett esophagus. Given that Barrett esophagus is itself a metaplastic mucosa and always has a certain degree of ‘‘baseline atypia’’ found in the glands in the lower portion of the mucosa, distinguishing this type of baseline atypia from BCDA with surface maturation can be quite challenging and undoubtedly is also subject to significant interobserver variation. Thus, reproducibility studies evaluating BCDA would be important to determine if this is a reproducible finding. As suggested by these authors, the findings by Lomo et al certainly warrant further investigation of this phenomenon as a possible subtype of Barrett esophagus-related dysplasia.

Development of the Mucosal Vascular System in the Distal Colon of the Fetal Mouse

Abstract: The formation of the crypt in the distal colon of the mouse was investigated in association with the development of vascular networks. For histological observation, 1-μm cross-sections were made from the distal colon of fetal mice in 13 to 18 days of gestation. Three-dimensional distributions of vascular networks in the organ were observed after perfusing fetuses with rhodamine isothiocyanate-labeled gelatin and immunostaining for laminin to examine the boundary between the epithelium and the mesenchyme. At 13 days of gestation, the distal colon and its epithelium formed a cylindrical tube and a loose primary plexus of vessels was built in the mesenchyme. In the distal colon of 15 days of gestation, the caudal portion began to form the crypt and the vascular plexus built up from a few layers was situated apart from the boundary between the epithelium and the mesenchyme. As the development proceeded, the formation of the crypt occurred in the caudorostral direction. The developing crypt advanced into the vascular plexus, so that a few vessels situated in the mesenchyme between crypts. As the crypt elongated, these vessels formed a small plexus situated perpendicular to the primary plexus, while the primary plexus became monolayered and loosened. The new plexus was composed of ascending vessels and traversing ones, but the regular honeycomb-like plexuses around openings of crypts have not established yet even in 18 days of gestation. The vascular system as well as the crypt in the distal colon will take further a few postnatal weeks to be completed. Anat Rec, 291:65–73, 2007. © 2007 Wiley-Liss, Inc.