The double stranded RNA analog poly-IC elicits both robust IFN-λ production and oncolytic activity in human gastrointestinal cancer cells.

PURPOSE: Type III IFN (IFN-λ) is the dominant frontline response over type I IFN in human normal intestinal epithelial cells upon viral infection, this response being mimicked by the dsRNA analog poly-IC. Poly-IC also induces cell death in murine intestinal crypts ex vivo. Here we examined whether these innate defense functions of normal intestinal epithelial cells are recapitulated in gastrointestinal carcinoma cells so that they could be harnessed to exert both immunoadjuvant and oncolytic functions, an unknown issue yet. EXPERIMENTAL DESIGN: Four human gastrointestinal carcinoma cell lines versus the Jurkat lymphoma cell line were used to assess the effects of intracellular poly-IC on i) IFN-λ secretion and cell proliferation and ii) role of NFκB signaling using the NFκB inhibitory peptide SN50 as a screening probe and a siRNA approach. RESULTS: Poly-IC induced in all cell lines except Jurkat both a robust IFN-λ secretion and a cytoreductive effect on adherent cells, restricted to proliferating cells and associated with cellular shedding and reduced clonogenicity of the shed cells. Collectively these findings demonstrate the oncolytic activity of poly-IC. Inhibiting NFκB in T84 cells using a siRNA approach decreased IFN-λ production without protecting the cells from the poly-IC oncolytic effects. In line with these findings IFN-λ, that upregulated the anti-viral protein MxA, was unable per se to alter T84 cell proliferation. CONCLUSION: Our demonstration that poly-IC-induced concomitant recapitulation of two innate functions of normal intestine, i.e. IFN-λ production and cell death, by human gastrointestinal cancer cells opens new perspectives in gastrointestinal cancer treatment.

Mapping clinicopathological entities within colorectal mucinous adenocarcinomas: a hierarchical clustering approach.

The aim of this study was to interrogate the heterogeneity of colorectal mucinous adenocarcinomas. This study is based on hierarchical clustering approach combining clinicopathological and molecular patterns known to be relevant to oncogenesis and therapeutic management of patients with colorectal carcinoma, ie, microsatellite instability, O6-methylguanine-DNA methyltransferase (MGMT) status, KRAS, and BRAF mutations and wnt signaling pathway activation. Comparison of the study group of 60 mucinous adenocarcinomas defined according to World Health Organization classification with control group of 136 colorectal adenocarcinomas successively removed shows higher frequency of BRAF and KRAS mutations and microsatellite instability-high status and lower frequency of wnt signaling pathway activation in mucinous adenocarcinomas. Hierarchical clustering isolated three relevant clusters: (i) cluster of microsatellite stable mucinous adenocarcinomas (54%) with KRAS mutation, and frequent MGMT changes, more frequently located in the left colon, often associated with contiguous precursor adenoma; (ii) cluster of BRAF-mutated mucinous adenocarcinomas (28%) with either microsatellite instability-high or microsatellite stable status, occurring in elderly female patients, nearly all located in the right colon, having the signature of serrated pathway of carcinomas; and (iii) a heterogeneous cluster of microsatellite instability-high mucinous carcinomas (18%), including inherited colorectal carcinomas, displaying a high-grade histological pattern. Age, TNM stage, and BRAF mutation had prognostic value. Hierarchical clustering analysis led to the identification of several clinicopathological entities of colorectal mucinous adenocarcinomas with epidemiologic, prognostic, and therapy relevance. Both KRAS and BRAF mutations appear as drivers in the alternate oncogenetic pathways governing the development of sporadic colorectal mucinous adenocarcinomas.

Interferon-Alpha Promotes Th1 Response and Epithelial Apoptosis via Inflammasome Activation in Human Intestinal Mucosa.

BACKGOUND & AIMS: Several lines of investigation suggest that interferon (IFN) alpha can alter human intestinal mucosa homeostasis. These include the endogenous production of IFN alpha in celiac disease or inflammatory bowel diseases, as well as the occurrence of intestinal side effects of exogenous IFN alpha used as a therapeutic tool. Here, we present an ex vivo translational approach to investigate the effects of IFN alpha on the human normal intestinal mucosa, as well as its underlying mechanisms. METHODS: Human normal colonic mucosa explants were cultured in the presence or absence of IFN alpha 2a. Epithelial homeostasis was assessed using the immunohistochemical marker of apoptosis M30. The Wnt inhibitor Dickkopf-Homolog-1 (DKK1) was assayed in the supernatants by enzyme-linked immunosorbent assay. Activation of the inflammasome (caspase-1/interleukin [IL]18) and of a Th1 response was determined by in situ detection of active caspase-1, as well as by measurement of mature IL18 production and the prototype Th1 cytokine IFN gamma by enzyme-linked immunosorbent assay. In addition, mechanistic studies were performed using the specific caspase-1 inhibitor Tyr-Val-Ala-Asp(OMe)-fluoromethylketone (YVAD-FMK), IL18-binding protein, neutralizing anti-IFN gamma, and anti-DKK1 antibodies. RESULTS: IFN alpha 2a elicited a rapid (24 hours) disruption of surface and crypt colonic epithelial cells via apoptosis that was variable in intensity among the 20 individuals studied. This apoptotic effect was dependent on the initiation of an IFN gamma response elicited by resident T box expressed in T cells-positive lamina propria cells. Both apoptosis and Th1 response were subordinated to active caspase-1 and IL18 production. Finally, neutralization of IFN gamma-induced DKK1 partially protected against IFN alpha-induced epithelial apoptosis. CONCLUSIONS: By using an ex vivo model, we show an interindividual heterogeneity of IFN alpha effects. We show that IFN alpha is able to disrupt both epithelial and immune homeostasis in the human intestine, by activation of an innate immunity platform, the inflammasome, which drives a Th1 response and leads to epithelial barrier disruption.

Notch signaling mediates crosstalk between endothelial cells and macrophages via Dll4 and IL6 in cardiac microvascular inflammation.

Although short-term outcomes have improved with modern era immunosuppression, little progress has been made in long-term graft survival in cardiac transplantation. Antibody-mediated rejection (AMR) is one of the leading causes of graft failure and contributes significantly to poor long-term outcomes. Endothelial cell (EC) injury, intravascular macrophage infiltrate and microvascular inflammation are the histological features of AMR. Nevertheless, mechanisms of AMR remain unclear and treatment is still limited. Here, we investigated the mechanisms underlying vascular and inflammatory cell network involved in AMR at endothelial and macrophage levels, using endomyocardial transplant biopsies and EC/monocyte cocultures. First, we found that AMR associates with changes in Notch signaling at endothelium/monocyte interface including loss of endothelial Notch4 and the acquisition of the Notch ligand Dll4 in both cell types. We showed that endothelial Dll4 induces macrophage polarization into a pro-inflammatory fate (CD40(high)CD64(high)CD200R(low) HLA-DR(low)CD11b(low)) eliciting the production of IL-6. Dll4 and IL-6 are both Notch-dependent and are required for macrophage polarization through selective down and upregulation of M2- and M1-type markers, respectively. Overall, these findings highlight the impact of the graft's endothelium on macrophage recruitment and differentiation upon AMR via Notch signaling. We identified Dll4 and IL-6 as coregulators of vascular inflammation in cardiac transplantation and as potential targets for immunotherapy.

Expanding the clinical spectrum of hereditary fibrosing poikiloderma with tendon contractures, myopathy and pulmonary fibrosis due to FAM111B mutations.

BACKGROUND: Hereditary Fibrosing Poikiloderma (HFP) with tendon contractures, myopathy and pulmonary fibrosis (POIKTMP [MIM 615704]) is a very recently described entity of syndromic inherited poikiloderma. Previously by using whole exome sequencing in five families, we identified the causative gene, FAM111B (NM_198947.3), the function of which is still unknown. Our objective in this study was to better define the specific features of POIKTMP through a larger series of patients. METHODS: Clinical and molecular data of two families and eight independent sporadic cases, including six new cases, were collected. RESULTS: Key features consist of: (i) early-onset poikiloderma, hypotrichosis and hypohidrosis; (ii) multiple contractures, in particular triceps surae muscle contractures; (iii) diffuse progressive muscular weakness; (iv) pulmonary fibrosis in adulthood and (v) other features including exocrine pancreatic insufficiency, liver impairment and growth retardation. Muscle magnetic resonance imaging was informative and showed muscle atrophy and fatty infiltration. Histological examination of skeletal muscle revealed extensive fibroadipose tissue infiltration. Microscopy of the skin showed a scleroderma-like aspect with fibrosis and alterations of the elastic network. FAM111B gene analysis identified five different missense variants (two recurrent mutations were found respectively in three and four independent families). All the mutations were predicted to localize in the trypsin-like cysteine/serine peptidase domain of the protein. We suggest gain-of-function or dominant-negative mutations resulting in FAM111B enzymatic activity changes. CONCLUSIONS: HFP with tendon contractures, myopathy and pulmonary fibrosis, is a multisystemic disorder due to autosomal dominant FAM111B mutations. Future functional studies will help in understanding the specific pathological process of this fibrosing disorder.

Heterogeneity of subordination of the IL-18/IFN-γ axis to caspase-1 among patients with Crohn's disease.

In Crohn's disease (CD), hierarchical architecture of the inflammatory network, including subordination of IL-18, an IFN-γ-inducing cytokine, to the inflammasome, have remained undeciphered. Heterogeneity among patients of such a subordination cannot be evaluated by animal models, monofactorial in their etiology and homogenous in disease progression. To address these issues, we set up an ex vivo model of inflamed mucosa explant cultures from patients with active long-standing CD. Th1 cytokine production, especially IFN-γ and IL-18, was assessed in relation with inflammation intensity. Subordination of the Th1 response to caspase-1, effector of the inflammasome, was determined in explant cultures subjected to pharmacological inhibition of caspase-1 by YVAD. We showed a correlation between secreted IFN-γ/IL-18 levels, and caspase-1 activation, with inflammation intensity of intestinal CD mucosa explants. Inhibition of caspase-1 activation using the specific inhibitor YVAD identified a homogenous non responder group featuring a caspase-1-independent IL-18/IFN-γ response, and a heterogenous responder group, in which both IL-18 and IFN-γ responses were caspase-1-dependent, with a 40-70% range of inhibition by YVAD. These findings bring out the concept of heterogeneity of subordination of the Th1 response to inflammasome activation among CD patients. This ex vivo model should have therapeutic relevance in allowing to determine eligibility of CD patients for new targeted therapies.

Hierarchical clustering identifies a subgroup of colonic adenocarcinomas expressing crypt-like differentiation markers, associated with MSS status and better prognosis.

The aim of this study was to identify in the group of colonic adenocarcinomas, not otherwise specified (NOS), subgroups of oncogenetic and prognostic significance based on the expression of immunohistochemical markers of epithelial cell differentiation of the gastrointestinal tract. Hierarchical clustering analysis of 122 adenocarcinomas (NOS) identified four clusters based on how closely their profile of immunohistochemical expression of differentiation markers was related: (i) a major cluster of 83 adenocarcinomas (68%) called crypt-like carcinoma (CLA) with a immunohistochemically expressing colonic crypt differentiation markers (cytokeratin 20+, CDX2+, MUC2+ or MUC2-) and (ii) three minor clusters, characterized by the loss of colonic crypt differentiation markers and/or the acquisition of expression of markers of metaplastic foveolar gastric differentiation (MUC5AC+) and/or aberrant cytokeratin 7 expression. CLAs were invariably MSS (χ (2) test: p < 0.0001). The sole parameters associated with worse overall survival of the 122 patients with adenocarcinoma (NOS) were pT stage, pN+ stage, and advanced clinical stage. Interestingly, CLA lineage of differentiation was an independent prognostic parameter for better overall survival among the 40 patients with an adenocarcinoma (NOS) stage III. In conclusion, hierarchical clustering led to the identification of a main cluster of adenocarcinoma (NOS) with crypt-like differentiation, associated with MSS status and better prognosis. Its value as a biomarker of response to conventional chemotherapeutic agents deserves to be examined in randomized therapy trials.

Acute cytotoxicity of MIRA-1/NSC19630, a mutant p53-reactivating small molecule, against human normal and cancer cells via a caspase-9-dependent apoptosis.

Although numerous studies have focused on the mechanisms of action of the candidate chemotherapeutic drug MIRA-1/NSC19630, initially described as a mutant p53-reactivating small molecule, the issue of its toxicological evaluation remains open. Here, we devised a strategy to examine the effects of MIRA-1 on a variety of human normal cells and cancer cell lines. First, we demonstrated a massive and rapid (within 2 hours) MIRA-1 apoptotic effect on human normal primary epithelial cells as shown using an intestinal mucosa explant assay. MIRA-1 was also cytotoxic to primary and subcultured human mesenchymal cells. Interestingly these effects were restricted to actively proliferating cells. Second, MIRA-1 acute toxicity was independent of p53, since it occurred in human normal cells with increased or silenced p53 expression level, in cancer cells derived from solid or liquid tumors, with either mutated or wt TP53, and in cancer cells devoid of p53. Third, combined pharmacological and genetic approaches showed that MIRA-1 acute cytotoxicity was mediated by a caspase-9-dependent apoptosis. In conclusion, our strategy unveils the limitations of the targeted action of a small molecule designed to reactivate mutant p53.

Reappraisal of the so-called 'villous tumours' of the rectosigmoid, based on histological, immunohistochemical and genotypic features.

BACKGROUND: Villous tumours of the rectosigmoid are historically defined as broad-based lesions associated with secretory diarrhoea. OBJECTIVE: This study aimed to perform a reappraisal of these tumours, on the basis of newly introduced histological, immunohistochemical and molecular parameters. METHODS: For this study, 22 villous tumours, diagnosed by endoscopic criteria (19 Paris 0-IIa, three Paris 0-Is), were evaluated according to WHO classification. Microsatellite instability status, KRAS and BRAF mutations, MGMT status of villous tumours and associated invasive carcinoma were determined. RESULTS: The 22 villous tumours fell into four groups: 1) nine villous adenomas, 2) six tubulovillous adenomas, 3) three filiform traditional serrated adenomas, and 4) four traditional serrated adenomas with conventional dysplasia. Filiform serrated adenomas displayed a distinctive endoscopic protruding pattern (Paris 0-Is). Villous adenomas were strongly associated with secretory diarrhoea. All the villous tumours were microsatellite stable. Five tumours exhibited MGMT abnormalities. KRAS mutations were frequent in villous adenomas, whereas BRAF mutations were essentially detected in serrated lesions. Invasive carcinomas (n = 7) maintained the histopathological and molecular imprint of the prior villous tumour. CONCLUSION: The rectosigmoid villous tumours are histologically and molecularly heterogeneous, including serrated neoplasias. Endoscopic and clinical findings are predictive of the histopathological diagnosis of some of these distinct entities.

The disintegrin and metalloproteinase ADAM10 mediates a canonical Notch-dependent regulation of IL-6 through Dll4 in human endothelial cells.

Although the involvement of the disintegrin and metalloproteinase ADAM10 in several areas of vascular biology is now clearly established, its role in vascular inflammation and in Notch signaling at the endothelial level remains unclear. In this study, we demonstrated that ADAM10 specifically localizes in the CD31(+) endothelial cells (ECs) in normal human cardiac tissues and in cultured primary arterial ECs. In vitro, ADAM10 drives a specific regulation of the Notch pathway in vascular ECs. Using an ADAM10 gain and loss of function approach we show an ADAM10-dependent regulation of Dll1 and Dll4 expression in association with changes in Hes1 and Hey1 expression. We also identified IL-6, IL-8, MCP-1 and sVCAM-1 as novel targets of ADAM10 upon inflammation. Although Notch pathway does not seem to be required for the production of IL-8, MCP-1 and sVCAM-1, the release of IL-6 by ECs occurred through ADAM10 and a canonical Notch signaling pathway, dependent of γ-secretase activity. Moreover, sustained expression of Dll4 mediated by ADAM10 elicits an increased release of IL-6 suggesting a strong implication of the specific Dll4 signaling in this mechanism. Modulation of IL-6 mediated by ADAM10/Notch signaling required PI3K activity. Thus, our findings suggest that ADAM10/Dll4 signaling is a major signaling pathway in ECs driving inflammatory events involved in inflammation and immune cell recruitment.

Association of HER1 amplification with poor prognosis in well differentiated gastric carcinomas.

AIMS: The pattern of E-cadherin expression and the HER1/HER2 status were studied in European patients with gastric carcinomas in relation with their differentiation and prognosis. METHODS: 82 gastric carcinomas (five papillary, 52 tubular, 19 poorly cohesive and six mixed according to WHO classification) were investigated for E-cadherin distribution (normal: restricted to the membrane; abnormal: absent or cytoplasmic expression), HER1 and HER2 expression using HercepTest and amplification using fluorescent in situ hybridisation. Statistical analysis assessed the association between the markers and their correlation with clinicopathological parameters and follow-up information. RESULTS: Abnormal E-cadherin distribution was found in 34 of the 82 gastric carcinomas (41%) (18/25 poorly cohesive or mixed (72%); 16/57 papillary or tubular type (28%)). HER1 overexpression (3+) and equivocal expression (2+) were found in five carcinomas (6%; four tubular and one poorly cohesive) and eight carcinomas (10%; six tubular and two poorly cohesive), respectively. HER2 overexpression (3+) and equivocal expression (2+) were found in seven carcinomas (8%; five papillary and two tubular) and three carcinomas (4%; three tubular), respectively. Amplification of HER1 or HER2 was detected in 14 gastric carcinomas (five papillary and nine tubular). All of them showed a normal E-cadherin distribution. In the univariate analysis, only HER1 amplification had a prognostic impact, while HER2 amplification and E-cadherin expression/distribution were not per se prognostically relevant. CONCLUSIONS: E-cadherin immunostaining and HER1 in situ hybridisation define a group of well differentiated gastric carcinomas with poor prognosis eligible for an aggressive therapeutic approach.

Mutations in FAM111B cause hereditary fibrosing poikiloderma with tendon contracture, myopathy, and pulmonary fibrosis.

Congenital poikiloderma is characterized by a combination of mottled pigmentation, telangiectasia, and epidermal atrophy in the first few months of life. We have previously described a South African European-descent family affected by a rare autosomal-dominant form of hereditary fibrosing poikiloderma accompanied by tendon contracture, myopathy, and pulmonary fibrosis. Here, we report the identification of causative mutations in FAM111B by whole-exome sequencing. In total, three FAM111B missense mutations were identified in five kindreds of different ethnic backgrounds. The mutation segregated with the disease in one large pedigree, and mutations were de novo in two other pedigrees. All three mutations were absent from public databases and were not observed on Sanger sequencing of 388 ethnically matched control subjects. The three single-nucleotide mutations code for amino acid changes that are clustered within a putative trypsin-like cysteine/serine peptidase domain of FAM111B. These findings provide evidence of the involvement of FAM111B in congenital poikiloderma and multisystem fibrosis.

Differential roles of Hath1, MUC2 and P27Kip1 in relation with gamma-secretase inhibition in human colonic carcinomas: a translational study.

Hath1, a bHLH transcription factor negatively regulated by the γ-secretase-dependent Notch pathway, is required for intestinal secretory cell differentiation. Our aim was fourfold: 1) determine whether Hath1 is able to alter the phenotype of colon cancer cells that are committed to a differentiated phenotype, 2) determine whether the Hath1-dependent alteration of differentiation is coupled to a restriction of anchorage-dependent growth, 3) decipher the respective roles of three putative tumor suppressor genes Hath1, MUC2 and P27kip1 in this coupling and, 4) examine how our findings translate to primary tumors. Human colon carcinoma cell lines that differentiate along a mucin secreting (MUC2/MUC5AC) and/or enterocytic (DPPIV) lineages were maintained on inserts with or without a γ-secretase inhibitor (DBZ). Then the cells were detached and their ability to survive/proliferate in the absence of substratum was assessed. γ-secretase inhibition led to a Hath1-mediated preferential induction of MUC2 over MUC5AC, without DPPIV modification, in association with a decrease in anchorage-independent growth. While P27kip1 silencing relieved the cells from the Hath1-induced decrease of anchorage-independent growth, MUC2 silencing did not modify this parameter. Hath1 ectopic expression in the Hath1 negative enterocytic Caco2 cells led to a decreased anchorage-independent growth in a P27kip1-independent manner. In cultured primary human colon carcinomas, Hath1 was up-regulated in 7 out of 10 tumors upon DBZ treatment. Parallel MUC2 up-regulation occurred in 4 (4/7) and P27kip1 in only 2 (2/7) tumors. Interestingly, the response patterns of primary tumors to DBZ fitted with the hierarchical model of divergent signalling derived from our findings on cell lines.

A spontaneous mutation of the rat Themis gene leads to impaired function of regulatory T cells linked to inflammatory bowel disease.

Spontaneous or chemically induced germline mutations, which lead to Mendelian phenotypes, are powerful tools to discover new genes and their functions. Here, we report an autosomal recessive mutation that occurred spontaneously in a Brown-Norway (BN) rat colony and was identified as causing marked T cell lymphopenia. This mutation was stabilized in a new rat strain, named BN(m) for "BN mutated." In BN(m) rats, we found that the T cell lymphopenia originated in the thymus, was intrinsic to CD4 T lymphocytes, and was associated with the development of an inflammatory bowel disease. Furthermore, we demonstrate that the suppressive activity of both peripheral and thymic CD4(+) CD25(bright) regulatory T cells (Treg) is defective in BN(m) rats. Complementation of mutant animals with BN Treg decreases disease incidence and severity, thus suggesting that the impaired Treg function is involved in the development of inflammatory bowel disease in BN(m) rats. Moreover, the cytokine profile of effector CD4 T cells is skewed toward Th2 and Th17 phenotypes in BN(m) rats. Linkage analysis and genetic dissection of the CD4 T cell lymphopenia in rats issued from BN(m)×DA crosses allowed the localization of the mutation on chromosome 1, within a 1.5 megabase interval. Gene expression and sequencing studies identified a frameshift mutation caused by a four-nucleotide insertion in the Themis gene, leading to its disruption. This result is the first to link Themis to the suppressive function of Treg and to suggest that, in Themis-deficient animals, defect of this function is involved in intestinal inflammation. Thus, this study highlights the importance of Themis as a new target gene that could participate in the pathogenesis of immune diseases characterized by chronic inflammation resulting from a defect in the Treg compartment.

Delineation of the infrequent mosaicism of KRAS mutational status in metastatic colorectal adenocarcinomas.

This study addresses the extent of the heterogeneity of KRAS status, present in a minority of metastatic colorectal carcinomas (mCRCs), on the basis of a thorough analysis of surgical resection specimens. Eighteen patients with mCRC were included. KRAS mutations (exon 2, codons 12 and 13) were determined using PCR and subsequent direct sequencing. This analysis included primary tumours (n=21), synchronous (n=10) and metachronous (n=18) matched metastases, and pelvic recurrence (n=1). Heterogeneity of KRAS status consisted in KRAS mutated in (i) the primary tumour but not in its synchronous metastasis, (ii) the metastasis but not in the primary tumour, (iii) the pelvic recurrence but not in the primary tumour, (iiii) some metastases and not in others from the same patient. Finally, the KRAS status varied among different areas of the same metastatic focus. This study defines the concept of KRAS mosaicism that affects a minority of mCRCs.

HLA-E/ß2 microglobulin overexpression in colorectal cancer is associated with recruitment of inhibitory immune cells and tumor progression.

The host immune response plays a major role in colorectal carcinoma (CRC) progression. A mechanism of tumor immune escape might involve expression of the human leucocyte antigen (HLA)-E/ß2m on tumor cells. The inhibitory effect of HLA-E/ß2m on CD8+ cytotoxic T lymphocytes and natural killer (NK) cells is mediated by the main HLA-E receptor CD94/NKG2A. As the pathophysiological relevance of this mechanism in CRC remains unknown, this prompted us to examine, in situ, in a series of 80 CRC (i) the HLA-E and ß2m coexpression by tumor cells, (ii) the density of CD8+, cytotoxic, CD244+ and NKP46+ intraepithelial tumor-infiltrating lymphocyte (IEL-TIL) and (iii) the expression of CD94/NKG2 receptor on IEL-TIL. These data were then correlated to patient survival. We provided (i) the in situ demonstration of HLA-E/ß2m overexpression by tumor cells in 21% of CRC characterized by an overrepresentation of signet ring cell carcinomas, mucinous carcinomas and medullary carcinomas, (ii) the significant association between HLA-E/ß2m overexpression by tumor cells and increased density of CD8+ cytotoxic, CD244+ and CD94+ IEL-TIL and (iii) finally, the unfavorable prognosis associated with HLA-E/ß2m overexpression by tumor cells. Our findings show that HLA-E/ß2m overexpression is a surrogate marker of poor prognosis and point to a novel mechanism of tumor immune escape in CRC in restraining inhibitory IEL-TIL.

A multiparametric approach to monitor the effects of γ-secretase inhibition along the whole intestinal tract.

γ-secretase inhibitors (GSIs) have been recently proposed as chemopreventive agents in gastrointestinal neoplasia, because they lead, through inhibition of the Notch signaling pathway, to goblet cell conversion in some intestinal adenomas of the Apc(Min) mice, and halt epithelial cell proliferation. In this study, we examine in depth, in normal mice, the effects of a GSI, dibenzazepine (DBZ), intraperitoneally administered for 8 days at a non toxic dose, on the gene expression pattern of secretory mucin (MUC), goblet cell conversion, organization of the crypt structural-proliferative units, stem cell niche and apoptotic compartments, along the entire length of the small intestine and colon. We demonstrate that DBZ elicits a homogeneous goblet cell conversion all along the mouse intestinal tract, associated with an overexpression of the gene Muc2 without ectopic expression of the gastric genes Muc5ac and Muc6, and with the emergence of lysozyme-positive 'intermediate cells' in the colon. Furthermore, DBZ treatment induces a heterogeneous reorganization of the crypt structural-proliferative units along the intestinal tract and of the stem cell niche in the colon, without disturbing the apoptotic compartment. These findings point to uncoupled effects of a GSI on goblet cell conversion and reorganization of the intestinal crypt structural-proliferative units and stem cell niche, and suggest caution in the use of GSIs as chemopreventive agents for intestinal neoplasia.

ADAM15 to α5ß1 integrin switch in colon carcinoma cells: a late event in cancer progression associated with tumor dedifferentiation and poor prognosis.

ADAM15, a member of the A Disintegrin And Metalloproteinase (ADAM) family, is a membrane protein containing an adhesion domain that binds to α5ß1 integrin through a unique RGD domain. ADAM15, expressed by human normal colonocytes, is involved in epithelial wound healing and tissue remodeling in inflammatory bowel disease. The aims of our study were (i) to analyze ADAM15 expression in a series of colon carcinomas and paired normal mucosa and (ii) to integrate the spatial relationship of ADAM15 with its binding partners α5ß1 integrin, a mesenchymal marker, as well as with other adhesion molecules, α3ß1 integrin and E-cadherin. A series of 94 colon carcinomas of the non other specified category were graded according to the World Health Organization classification. Immunohistochemistry was performed on frozen tissue sections using antibodies directed to ADAM15, α5ß1 and α3ß1 integrins, and E-cadherin. ADAM15 was quantified at the mRNA level. Finally, promoter methylation of ADAM15 was examined as well as the microsatellite instability status (MSS/MSI). Thirty-six percent of colorectal carcinomas displayed a reduced expression of ADAM15 in cancer cells, confirmed at the mRNA level in most cases, without promoter methylation. ADAM15 down-regulation was associated with histologically poorly differentiated carcinomas. In addition, it was associated with the acquisition of α5ß1 by cancer cells and down-regulation of α3ß1 integrin and E-cadherin. Finally this profile that includes characteristic of epithelial to mesenchymal transition is a late progression event of colon cancer with a poor prognosis.

Loss of interleukin-10 or transforming growth factor ß signaling in the human colon initiates a T-helper 1 response via distinct pathways.

BACKGROUND & AIMS: Signaling via interleukin (IL)-10 or transforming growth factor (TGF)-ß is disrupted in subpopulations of patients with inflammatory bowel disease, but it is not clear how a T-helper (Th) 1 cell response is induced. We studied conversion of human mucosal innate immune cells into inflammatory cells and the initiation of a Th1 cell response following loss of IL-10 or TGF-ß signaling. METHODS: We depleted IL-10 or TGF-ß from explant cultures of human normal colonic mucosa using immunoneutralization. Pharmacologic inhibitors and antibodies were used to determine the factors involved in the initiation of an interferon (IFN)-γ response following loss of TGF-ß or IL-10 signaling. Cytokines produced by mucosal cells were assessed by enzyme-linked immunosorbent assay and quantitative reverse-transcriptase polymerase chain reaction. The subsets of cells involved in cytokine production were determined by in situ immunofluorescence analysis and flow cytometry after digestion of the explants with collagenase. RESULTS: Depletion of IL-10 from human normal colonic mucosa resulted in an IFN-γ response, characterized by early-stage secretion of mature IL-18 and production of the active form of caspase-1 by macrophages and some epithelial cells. A caspase-1 inhibitor or the IL-18 antagonist IL-18-binding protein blocked this response. By contrast, depletion of TGF-ß resulted in an IFN-γ response that was preceded by and required secretion of IL-12 from macrophages, dendritic cells, and epithelial cells. CONCLUSIONS: Innate immune cells (macrophages and epithelial cells) activate a Th1 cell response in explant cultures of human normal colonic mucosa depleted in IL-10 or TGF-ß via distinct, nonredundant pathways. These pathways might contribute to the pathogenesis of inflammatory bowel disease.

Time-resolved release of calcium from an epithelial cell monolayer during mucin secretion.

A significant amount of Ca²+ is contained in secretory mucin granules. Exchange of Ca²+ for monovalent cations drives the process of mucin decondensation and hydration after fusion of granules with the plasma membrane. Here we report direct observation of calcium secretion with a Ca²+ ion-selective electrode (ISE) in response to apical stimulation with ATP from HT29-Cl.16E cells, a subclone of the human colonic cancer cell line HT29. No increase in Ca²+ level was seen for the sister cell line Cl.19A, which lacks mucin granules, or for Cl.16E cells after inhibition of granule fusion with wortmannin. Further, the measured concentration was used to estimate the time-resolved rate of release of Ca²+ from the cell monolayer, by use of a deconvolution-based method developed previously (Nair and Gratzl in Anal Chem 77:2875-2881, 2005). The results argue that Ca²+ release by Cl.16E cells is associated specifically with mucin secretion, i.e., that the measured Ca²+ increase in the apical solution is derived from granules after fusion and mucin exocytosis. The Ca²+ ISE in conjunction with deconvolution provides a minimally disturbing method for assessment of Ca²+ secretion rates. The release rates provide estimates of exocytosis rates and, when combined with earlier capacitance measurements, estimates of post-stimulation endocytosis rates also.