Impact of high-fat feeding on basic helix-loop-helix transcription factors controlling enteroendocrine cell differentiation.

BACKGROUND AND OBJECTIVES: Gut hormones secreted by enteroendocrine cells (EECs) play a major role in energy regulation. Differentiation of EEC is controlled by the expression of basic helix-loop-helix (bHLH) transcription factors. High-fat (HF) feeding alters gut hormone levels; however, the impact of HF feeding on bHLH transcription factors in mediating EEC differentiation and subsequent gut hormone secretion and expression is not known. METHODS: Outbred Sprague-Dawley rats were maintained on chow or HF diet for 12 weeks. Gene and protein expression of intestinal bHLH transcription factors, combined with immunofluorescence studies, were analyzed for both groups in the small intestine and colon. Gut permeability, intestinal lipid and carbohydrate transporters as well as circulating levels and intestinal protein expression of gut peptides were determined. RESULTS: We showed that HF feeding resulted in hyperphagia and increased adiposity. HF-fed animals exhibited decreased expression of bHLH transcription factors controlling EEC differentiation (MATH1, NGN3, NEUROD1) and increased expression of bHLH factors modulating enterocyte expression. Furthermore, HF-fed animals had decreased number of total EECs and L-cells. This was accompanied by increased gut permeability and expression of lipid and carbohydrate transporters, and a decrease in circulating and intestinal gut hormone levels. CONCLUSIONS: Taken together, our results demonstrate that HF feeding caused decreased secretory lineage (that is, EECs) differentiation through downregulation of bHLH transcription factors, resulting in reduced EEC number and gut hormone levels. Thus, impaired EEC differentiation pathways by HF feeding may promote hyperphagia and subsequent obesity.

SCFAs strongly stimulate PYY production in human enteroendocrine cells.

Peptide-YY (PYY) and Glucagon-Like Peptide-1 (GLP-1) play important roles in the regulation of food intake and insulin secretion, and are of translational interest in the field of obesity and diabetes. PYY production is highest in enteroendocrine cells located in the distal intestine, mirroring the sites where high concentrations of short chain fatty acids (SCFAs) are produced by gut microbiota. We show here that propionate and butyrate strongly increased expression of PYY but not GCG in human cell line and intestinal primary culture models. The effect was predominantly attributable to the histone deacetylase inhibitory activity of SCFA and minor, but significant contributions of FFA2 (GPR43). Consistent with the SCFA-dependent elevation of PYY gene expression, we also observed increased basal and stimulated PYY hormone secretion. Interestingly, the transcriptional stimulation of PYY was specific to human-derived cell models and not reproduced in murine primary cultures. This is likely due to substantial differences in PYY gene structure between mouse and human. In summary, this study revealed a strong regulation of PYY production by SCFA that was evident in humans but not mice, and suggests that high fibre diets elevate plasma concentrations of the anorexigenic hormone PYY, both by targeting gene expression and hormone secretion.

Immune function of patients with gastrointestinal carcinoma after treatment with multiple infusions of monoclonal antibody 17.1A.

Ten patients with metastatic adenocarcinomas of the colon or pancreas were treated with multiple injections of monoclonal antibody 17.1A. For each injection, antibody concentration in the patients' sera plateaued during the entire treatment course, and then decreased, with faster antibody clearance in patients given previous injections of mouse monoclonal antibodies for immunoscintigraphy. Six of the ten patients were able to generate anti-mouse antibodies, detectable 7 days after the initial infusion. Peripheral blood mononuclear cells from patients showed only low level ability to mediate spontaneous and antibody-dependent cytotoxicity in vitro, both before monoclonal antibody treatment and during the entire treatment period. Undiluted sera from these patients were unable to generate antibody-dependent cytotoxicity activity in vitro at any time during the observation period.

Immunoglobulin class and immunoglobulin G subclass analysis of human anti-mouse antibody response during monoclonal antibody treatment of cancer patients.

The immune response against murine IgG2a immunoglobulin was studied in 42 gastrointestinal cancer patients treated with mouse monoclonal antibodies. IgM, IgA, and all 4 IgG subclasses directed against mouse immunoglobulin were studied. Thirty of 42 patients developed an anti-mouse response of IgG isotype, often associated with IgM and IgA specific for mouse monoclonal antibodies circulating in serum. Patients first developed an IgM response, followed by IgA and IgG. IgG1 was found to be the most frequently expressed isotype, followed by IgG2 and IgG3. IgG4 was detected in only 6 patients.

Monoclonal antibodies to a rat colon carcinoma: model for monoclonal antibody therapy of solid tumors.

Tumor progression, lung metastasis, and death occur in tumor-bearing BD IX syngeneic rats in a fashion similar to the course of patients with metastatic colon cancer. In an effort to establish a relevant model for monoclonal antibody (MoAb) therapy of tumors, we generated murine MoAb against DHD/TR, a dimethylhydrazine-induced rat colon carcinoma which has been adapted to cell culture. Murine MoAb 17B10 E4 (E4) reacts with the TR tumor and shows weak immunoperoxidase reactivity with normal rat tissues. Murine MoAb 5F7 D3 (D3) reacts with the tumor and a variety of normal rat epithelia. Both are IgG2a and mediate cytotoxicity by rat peripheral blood mononuclear cells. 18D5 F6 (F6) also reacts with the tumor and normal tissues but is an IgG2b and does not mediate cytotoxicity in the presence of rat effector cells. Iodinated E4 and D3 antibodies retained their immunoreactivity. E4 revealed 9.8 x 10(5) antigenic sites per TR cell, with an affinity constant of 9.35 x 10(7) M-1, while D3 demonstrated 2.5 x 10(6) antigenic sites and an affinity constant of 4.2 x 10(7) M-1. Immunoblotting showed that the antigens recognized by D3 and E4 are glycoproteins with molecular weights of 27,000 and 66,000, respectively. F6 failed to react with its antigen present in the blot. This rat colon carcinoma and the monoclonal antibodies described here may provide experimental data useful for implementing monoclonal antibodies in cancer therapy.

A new tumor-associated antigen expressed on breast carcinomas, defined by monoclonal antibody BCA 227.

After immunization of mice with the human breast carcinoma cell line MCF-7, we produced monoclonal antibody (mAb) BCA 227, which allowed us to characterize a new tumor-associated antigen. This molecule is strongly expressed by well differentiated mammary carcinoma cell lines and by some other tumor cell lines of epithelial origin. Immunohistological study of frozen sections of different tissues and tumors confirmed its expression by tumor cells of epithelial origin, particularly infiltrating duct carcinomas of the breast. The antigen is also expressed, to a lesser extent, by some normal epithelial cells. Its biochemical characterization revealed a Mr 71,000 protein without an N-linked sugar moiety. Six to 40 x 10(3) binding sites are present on breast tumor cell surfaces. Although mAb BCA 227, which was found to be of the IgG2a isotype, did not mediate antibody-dependent cell-mediated cytotoxicity with either human or mouse effector cells, a 50% inhibition of SK-BR5 tumor growth was obtained in nude mice, suggesting that another mechanism is responsible for this inhibition. Biodistribution studies of radiolabeled F(ab')2 fragments of mAb BCA 227 in tumor-bearing nude mice showed a preferential localization in the tumor. All these data are in favor of the use of mAb BCA 227 as an immunodiagnostic tool for breast cancer.

Characterization of a human monocyte antigen, B148.4, regulated during cell differentiation and activation.

We analyzed the phenotypic changes associated with monocyte activation and differentiation using a newly developed monoclonal antibody (B148.4). Among peripheral blood leukocytes, the antigen recognized by this antibody is expressed on monocytes and granulocytes at high and low density, respectively. Antigen expression is lost during in vitro differentiation of monocytes and is absent on tissue macrophages, indicating that expression of this antigen is related to monocyte differentiation. Only 1 alpha,25-dihydroxyvitamin D3 and phorbol diesters, of several inducers tested, up-regulate B148.4 antigen expression on cells (monocytes and certain myeloid cell lines) that constitutively bear the antigen, without, however, allowing its maintenance during monocytic differentiation or inducing it on negative cells. By immunoprecipitation from B148.4+ U937 cells, the antigen is a complex of a major 116-kd and two minor 38- and 46-kd molecules. Analysis of eight different tissues reveals that the antigen is shared with endothelial cells. Biochemical characteristics, cellular distribution, and expression pattern on monocytes, myeloid cell lines, and AML cells upon culture with different stimuli indicate that B148.4 is a novel monocyte differentiation antigen.

Rise in cytosolic Ca2+ concentration induced by P2-purinoceptor activation in isolated myocytes from the rat gastrointestinal tract.

1. The changes in the free cytosolic Ca2+ concentration ([Ca2+]i) in response to agonists of P2-purinoceptors were studied in myocytes isolated from the longitudinal muscle layer of different regions of the rat gastrointestinal tract (stomach, jejunum, ileum, caecum and colon). [Ca2+]i was estimated by emission from the fluorescent dye, indo-1. 2. ATP and the P2Y-purinoceptor agonist, 2-methylthio-ATP (2-MeSATP), transiently increased [Ca2+]i in single myocytes from all segments of the gastrointestinal tract, whereas alpha,beta-methylene-ATP, a P2x-purinoceptor agonist, had no effect. 3. The rise in [Ca2+]i induced by ATP and 2-MeSATP was maintained in Ca(2+)-free solution but was abolished by depletion of the intracellular store with thapsigargin (1 microM). 4. Single myocytes from stomach, caecum and colon also responded to UTP by a transient increase in [Ca2+]i. 5. Individual myocytes responded to ATP, 2-McSATP and UTP in a nearly all-or-nothing manner. The increasing of agonist concentration enhanced the number of responding cells but did not increase the amplitude of the [Ca2+]i rise. 6. These results suggest that myocytes from the longitudinal layer of gastrointestinal muscle do not possess functional P2x-purinoceptors and that agonists of P2Y and P2U-purinoceptors induced a rise in [Ca2+]i, probably via an all-or-nothing mobilization of Ca2+ from intracellular stores.

Cyclo-oxygenase-2 over-expression in sporadic colorectal carcinoma without lymph node involvement.

BACKGROUND: Cyclo-oxygenase-2 over-expression has been reported in most advanced human colorectal cancers. AIMS: To assess the prevalence of cyclo-oxygenase-2 over-expression in non-advanced colorectal cancers, to investigate the correlation between cyclo-oxygenase-2 status and tumour clinicopathological features and molecular phenotype, and to determine the impact of cyclo-oxygenase-2 status on long-term clinical outcome. METHODS: Sixty-one patients who had undergone surgery for colorectal cancer without lymph node involvement were evaluated retrospectively. Cyclo-oxygenase-2 expression was determined by immunohistochemistry. The tumour replication error phenotype was assessed by amplification of the two microsatellites, BAT-25 and BAT-26. RESULTS: Thirty-six tumours were classified as cyclo-oxygenase-2 positive and 25 as cyclo-oxygenase-2 negative. No correlation was found between cyclo-oxygenase-2 over-expression and clinicopathological features or molecular phenotype. Cyclo-oxygenase-2 over-expression was an independent predictor of a poor prognosis. Indeed, the relative risk of tumour recurrence or death for patients with cyclo-oxygenase-2-positive tumours was 2.13 times that of patients with cyclo-oxygenase-2-negative tumours (P=0.008; 95% confidence interval, 1.22-3.73). This difference remained significant when post-operative deaths were censored in the multivariate analysis (P=0.014). CONCLUSION: Cyclo-oxygenase-2 over-expression is not associated with tumour phenotype, but is indicative of a poorer clinical outcome in patients with non-advanced colorectal carcinoma.

Monocyte activity in the presence of calcium phosphate activated by 1,25 (OH)2 VD3 and interferon-gamma.

The monocyte is an essential element in cellular immunity. Consequently, its role in the biocompatibility process is important. A model study of the degradation of bioactive ceramics (calcium phosphate) using human peripheral blood monocytes activated by 1,25 (OH)2 VD3 and interferon (IFN)-gamma was implemented. Activated monocytes cultivated on calcium phosphate tablets led to cellular morphological modifications and to changes in the number of nuclei (from d 2 to 14). IFN-gamma promoted adhesion, the appearance of cytoplasmic extensions and multinuclei. Cavity resorption activity on the material was observed simultaneously.

Utilization of activated U937 monocytic cells as a model to evaluate biocompatibility and biodegradation of synthetic calcium phosphate.

The use of calcium phosphate biomaterials as a bone substitute necessitates the use of normative biocompatibility and biodegradation techniques which must be fast, simple and reproducible. In the present study, we have developed an in vitro model to study and to compare different calcium phosphate ceramics. After activation with 1,25-dihydroxy-vitamin D3 and phorbol 12,13-dibutyrate, the monoblastic U937 cells became multinucleated, expressed tartrate-resistant acid phosphatase and several markers of monocyte/macrophage differentiation. Activated U937 cells did not express the vitronectin receptor (VNR) (as revealed using monoclonal antibodies 23C6 or 13C2) but around 25% of the cells were strongly reactive with 211D, a novel monoclonal antibody that recognizes an osteoclast-specific membrane antigenic determinant. These cells remain active/viable with hydroxyapatite (HA) or beta-tricalcium phosphate (beta-TCP) ceramics. In conclusion, activated U937 cells are good candidates to use in a normative in vitro method to evaluate new biomaterials.

Molecular mechanisms involved in the antiproliferative effect of two COX-2 inhibitors, nimesulide and NS-398, on colorectal cancer cell lines.

BACKGROUND: Cyclooxygenase (COX)-2 is up-regulated in most colorectal cancers. Chronic use of non-steroidal anti-inflammatory drugs, which target cyclooxygenases, have been shown to reduce the risk of these cancers. However, the mechanisms underlying this protective effect remain unclear. AIMS: The aim of our study was to characterize the effects of two COX-2 selective inhibitors, NS-398 and nimesulide, on colorectal cancer cell proliferation, and to describe the molecular mechanisms involved. MATERIALS AND METHODS: HT-29 and SW-1116 cell lines were cultured with either NS-398 or nimesulide. Cell proliferation was assessed by staining DNA with crystal violet. Cell cycle repartition and apoptosis were analysed by flow cytometry. The expression of COX-1 and COX-2. and of two cyclin dependent kinase inhibitors, p21Cip1 and p27Kip1, was analysed by Western blotting and RT-PCR. RESULTS: Both drugs dose-dependently inhibited cell proliferation and induced G1 cell cycle blockade. HT-29 cells were more sensitive to both drugs than SW-1116 cells. p21Cip1 and p27Kip1 were induced on both cell lines. Concomitant induction of p21Cip1 mRNA indicates transcriptional modulation, whereas induction of p27Kip1 only at the protein level suggests post-translational modulation. CONCLUSION: NS-398 and nimesulide inhibit colorectal cell proliferation through induction of p21Cip1 and p27Kip1.

[Characterization of autoantigen (p105) in a model of colonic adenocarcinoma in the rat]. / Caractérisation d'un autoantigène (p105) dans un modèle d'adénocarcinome colique chez le rat.

Sera from BDIX rats bearing the syngeneic colon tumors PROb or REGb were analysed by Western blotting in order to detect a possible humoral response against the grafted tumor. The PROb clone grows progressively in syngeneic hosts and metastasizes, whereas the REGb clone grows slowly and then is rejected. This model was developed by F Martin and his group in Dijon, France. We observed that rats bearing PROb tumors only develop an antibody response against a water-soluble protein of 105 kDa (p105) which is expressed by both tumor clones. This antibody response has never been detected in rats bearing REGb tumors. The antigen p105 was also expressed by normal adult colon as well as some other foetal or adult tissues. It is also present in extracts from several tumor cell lines including human colorectal carcinoma cell lines. Moreover, the titer of detected antibody at day 30 was inversely correlated with the survival of rats after tumor inoculation, suggesting a possible facilitating role of this antibody response.

Development of a monoclonal antibody against dentin phosphophoryn: a tool to study odontoblastic activity.

The role played by phosphophoryn, one of the major noncollagenous proteins of dentin extracellular matrix, in the mineralization process has not been fully characterized. The purpose of our work was to produce monoclonal antibodies (MAbs) against dentin phosphophoryn and to test their reactivity with primary culture of odontoblasts. Dentin phosphophoryn (DPP) was extracted after the mechanical dissociation of teeth and dialyzed against guanidine and EDTA solutions followed by CaC1(2) precipitation. These extracts were characterized by SDS-PAGE and staining with Coomassie blue and Stains-All. After immunization of mice with these extracts, we produced MAb 7G4, which reacted with dentin phosphophoryn as revealed by Western blot. MAb 7G4 reactivity was tested against a primary culture of pig odontoblasts, revealing filaments specifically stained by the anti-DPP antibody. This antibody will be of great interest to study the mineralization process and dental pulp reaction after capping with various calcium phosphate materials.

Monoclonal antibodies specific immunotherapy of gastrointestinal tumors.

Monoclonal antibody 17-1A showing cytotoxic properties to GI tract adenocarcinoma cells in vitro and mediating tumor growth inhibition in nude mice, was used as immunotherapeutic agent in 95 patients with various metastatic gastrointestinal adenocarcinomas. Several clinical trials were performed in patients with metastatic disease unaccessible to more conventional therapy of proven efficacy. Results of the different trials are reported here. Tolerance to monoclonal antibody infusion was excellent with minor side effects, except when combinations of several monoclonal antibodies were used. Three complete responses, five partial responses inferior to 50% and 24 stable diseases were noticed. A randomized trial is presently performed in high risk cancer patients with B2 or C colorectal carcinomas with 17-1A as adjuvant immunotherapeutic agent after surgery.

[Expression of the gene of cholecystokinin. Demonstration from duodenal biopsies in man]. / Expression du gène de la cholécystokinine. Mise en évidence à partir de biopsies duodénales chez l'homme.

Cholecystokinin (CCK) is a peptide released after feeding. Until now, CCK gene expression has been studied only on sacrified animals on mucosa scrapped off the duodenum. OBJECTIVE--The aim of this study was to assess CCK-mRNA detection on duodenal biopsy specimens in subjects undergoing gastrointestinal endoscopy. METHODS--Six biopsy specimens were taken from 6 healthy subjects, a) after a 12-h overnight fast and b) 6 or 12 h after a fat meal, and inducing a CCK release. CCK-mRNA was analyzed by Northern blot. RESULTS--Plasma CCK levels increased from basal levels of 0.5 +/- 0.1 pmole/L to 8.2 +/- 1.5 pmole/L 10 min after the meal. The fasting CCK-mRNA levels were 44.0 +/- 0.6% and the post-prandial levels increased to 74.0 +/- 6.0% at 6 h and decreased to 47.5 +/- 0.5% at 12 h. CONCLUSIONS--Detection of CCK gene expression in human duodenal biopsy specimens is feasible. Stimulation of CCK release after a meal is followed by an increase in CCK-mRNA in the duodenal mucosa.

Effects of short-chain fatty acids on gastrointestinal motility.

Besides their action on gut morphology and function, short-chain fatty acids (SCFAs), produced by bacterial fermentation of carbohydrates in the colon, influence gastrointestinal motility. As they are not present in the stomach and proximal small intestine, SCFAs do not directly affect motility of these segments. However, caecal infusion of SCFAs as well as colonic fermentation of lactulose induce a relaxation of the proximal stomach in humans, indicating that SCFAs can affect motility at a distance from their site of production. Moreover, this suggests that SCFAs may be involved in the so-called "ileocolonic brake', i.e. the inhibition of gastric emptying by nutrients reaching the ileo-colonic junction. In the terminal ileum, where their concentration may increase following a colo-ileal reflux, SCFAs stimulate contractions and shorten ileal emptying, which may protect ileal mucosa against the potentially harmful effects of the reflux of colonic contents. Although SCFAs are produced and concentrated in the colon, their action on motility of this organ is not clearly understood and may depend on concentration, molecular structure of the acids, responsiveness of the colonic segments and animal species. The mechanisms of action of SCFAs on gastrointestinal motility are not completely elucidated. They may involve systemic humoral and neural pathways as well as local reflexes and myogenic responses.