Novel markers of the human follicle-associated epithelium identified by genomic profiling and microdissection.

BACKGROUND & AIMS: Regulation of gene expression in the follicle-associated epithelium (FAE) over Peyer's patches is largely unknown. CCL20, a chemokine that recruits immature dendritic cells, is one of the few FAE-specific markers described so far. Lymphotoxin beta (LTalpha1beta2) expressed on the membrane of immune cells triggers CCL20 expression in enterocytes. In this study, we measured expression profiles of LTalpha1beta2-treated intestinal epithelial cells and selected CCL20 -coregulated genes to identify new FAE markers. METHODS: Genomic profiles of T84 and Caco-2 cell lines treated with either LTalpha1beta2, flagellin, or tumor necrosis factor alpha were measured using the Affymetrix GeneChip U133A. Clustering analysis was used to select CCL20 -coregulated genes, and laser dissection microscopy and real-time polymerase chain reaction on human biopsy specimens was used to assess the expression of the selected markers. RESULTS: Applying a 2-way analysis of variance, we identified regulated genes upon the different treatments. A subset of genes involved in inflammation and related to the nuclear factor kappaB pathway was coregulated with CCL20 . Among these genes, the antiapoptotic factor TNFAIP3 was highly expressed in the FAE. CCL23 , which was not coregulated in vitro with CCL20 , was also specifically expressed in the FAE. CONCLUSIONS: We have identified 2 novel human FAE specifically expressed genes. Most of the CCL20 -coregulated genes did not show FAE-specific expression, suggesting that other signaling pathways are critical to modulate FAE-specific gene expression.

Generation of reconstituted human plasma-derived secretory-like IgA and IgM and their protective effect on intestinal epithelium

Les muqueuses sont les membranes tapissant les cavités du corps, tel que le tube digestif, et sont en contact direct avec l'environnement extérieur. Ces surfaces subissent de nombreuses agressions pouvant être provoquées par des agents pathogènes (bactéries, toxines ou virus). Cela étant, les muqueuses sont munies de divers mécanismes de protection dont notamment deux protéines-clés permettant de neutraliser les agents pathogènes : les anticorps ou immunoglobulines sécrétoires A (SIgA) et M (SIgM). Ces anticorps sont, d'une part, fabriqués au niveau de la muqueuse sous forme d'IgA et IgM. Lorsqu'ils sont sécrétés dans l'intestin, ils se lient à une protéine appelée pièce sécrétoire et deviennent ainsi SIgA et SïgM. La présence de la pièce sécrétoire est essentielle pour que les anticorps puissent fonctionner au niveau de la muqueuse. D'autre part, ces anticorps sont également fabriqués dans d'autres parties du corps en général et se retrouvent dans le sang sous forme d'IgA et IgM Chez l'homme, des thérapies basées sur l'injection d'anticorps donnent de bons résultats depuis de nombreuses années notamment dans le traitement des infections. Bien qu'un certain nombre d'études ont montré le rôle protecteur des anticorps de type IgA et IgM, ceux-ci ne sont que rarement utilisés dans les thérapies actuelles. La principale raison de cette faible utilisation réside dans la production ou la purification des IgA/IgM ou SIgA/SIgM (la forme active au niveau des muqueuses) qui est difficile à réaliser à large échelle. Ainsi, le but de la thèse était (1) d'étudier la possibilité d'employer des IgA et des IgM provenant du sang humain pour générer des SIgA et SIgM et (2) de voir si ces anticorps reconstitués pouvaient neutraliser certains agents pathogènes au niveau des muqueuses. Tout d'abord, une analyse biochimique des IgA et des IgM issues du sang a été effectuée. Nous avons observé que ces anticorps avaient des caractéristiques similaires aux anticorps naturellement présents au niveau des muqueuses. De plus, nous avons confirmé que ces anticorps pouvaient être associés à une pièce sécrétoire produite en laboratoire pour ainsi donner des SIgA et SIgM reconstituées. Ensuite, la fonctionnalité des anticorps reconstitués a été testée grâce à un modèle de couche unique de cellules intestinales différenciées (monocouches) en laboratoire imitant la paroi de l'intestin. Ces monocouches ont été infectées par une bactérie pathogène, Shigella flexneri, responsable de la shigellose, une maladie qui provoque des diarrhées sanglantes chez l'homme. L'infection des monocouches par les bactéries seules ou combinées aux SIgA et SIgM reconstituées a été analysée. Nous avons observé que les dommages des cellules étaient moins importants lorsque les SIgA étaient présentes. Il apparaît que les SIgA neutralisent les bactéries en se fixant dessus, ce qui provoque leur agrégation, et diminuent l'inflammation des cellules. La protection s'est montrée encore plus efficace avec les SIgM. De plus, nous avons vu que les SIgA et SIgM pouvaient diminuer la sécrétion de facteurs nocifs produits par les bactéries. Utilisant le même modèle des monocouches, la fonctionnalité des IgA issues du sang humain a aussi été testée contre une toxine sécrétée par une bactérie appelée Clostridium diffìcile. Cette bactérie peut être présente naturellement dans l'intestin de personnes saines, cependant elle peut devenir pathogène dans certaines conditions et être à l'origine de diarrhées et d'inflammations de l'intestin via la sécrétion de toxines. Des préparations d'anticorps contenant une certaine proportion de SIgA reconstituées ont amené à une diminution des dommages et de l'inflammation des monocouches causés par la toxine. L'ensemble de ces résultats prometteurs, montrant que des SIgA et SIgM reconstituées peuvent protéger la paroi de l'intestin des infections bactériennes, nous conduisent à approfondir la recherche sur ces anticorps dans des modèles animaux. L'aboutissement de ce type de recherche permettrait de tester, par la suite, l'efficacité sur l'homme de traitements des infections des muqueuses par injection d'anticorps de type SIgA et SIgM reconstituées. Les muqueuses, telle que la muqueuse gastrointestinale, sont des surfaces constamment exposées à l'environnement et leur protection est garantie par une combinaison de barrières mécaniques, physicochimiques et immunologiques. Parmi les divers mécanismes de protection immunologiques, la réponse humorale spécifique joue un rôle prépondérant et est assurée par les immunoglobulines sécrétoires de type A (SIgA) et M (SIgM). Les thérapies basées sur l'administration d'IgG apportent d'importants bénéfices dans le domaine de la santé. Bien que des études sur les animaux aient montré que l'administration par voie muqueuse d'IgA polymérique (plgA) ou SIgA pouvaient protéger des infections, des IgA/SIgA n'ont été utilisées qu'occasionnellement dans les thérapies. De plus, des études précliniques et cliniques ont démontré que l'administration par voie systémique de préparations enrichies en IgM pouvait aussi protéger des infections. Cependant, l'administration par voie muqueuse d'IgM/SIgM purifiées n'a pas été examinée jusqu'à présent. La principale raison est que la purification ou là production des IgA/SIgA et IgM/SIgM est difficile à réaliser à large échelle. Le but de ce travail de thèse était d'examiner la possibilité d'associer des IgA et IgM polyclonals purifiées à partir du plasma humain avec une pièce sécrétoire recombinante humaine afin de générer des SIgA et SIgM reconstituées fonctionnelles. Tout d'abord, une analyse biochimique des IgA et IgM issues du plasma humain a été effectuée par buvardage de western et Chromatographie. Ces molécules avaient des caractéristiques biochimiques similaires à celles des immunoglobulines issues de la muqueuse. L'association entre plgA ou IgM issues du plasma humain et la pièce sécrétoire recombinante humaine a été confirmée, ainsi que la stoechiométrie 1:1 de l'association. Comme dans les conditions physiologiques, cette association permettait de retarder la dégradation des SIgA et SIgM reconstituées exposées à des protéases intestinales. Ensuite, la fonctionnalité et le mode d'action des IgA et IgM issues du plasma humain, ainsi que des SIgA et SIgM reconstituées, ont été explorés grâce à un modèle in vitro de monocouches de cellules intestinales épithéliales polarisées de type Caco-2, qui imite l'épithélium intestinal. Les monocouches ont été infectées par un pathogène entérique, Shigella flexneri, seul ou combiné aux immunoglobulines issues du plasma humain ou aux immunoglobulines sécrétoires reconstituées. Bien que les dommages des monocouches aient été retardés par les plgA et SIgA reconstituées, les IgM et SIgM reconstituées se sont montrées supérieures dans le maintien de l'intégrité des cellules. Une agrégation bactérienne et une diminution de l'inflammation des monocouches ont été observées avec les plgA et SIgA reconstituées. Ces effets étaient augmentés avec les IgM et SIgM reconstituées. De plus, il s'est révélé que les deux types d'immunoglobulines de type sécrétoire reconstituées agissaient directement sur la virulence des bactéries en réduisant leur sécrétion de facteurs de virulence. La fonctionnalité des IgA issues du plasma humain a aussi été testée contre la toxine A de Clostridium difficile grâce au même modèle de monocouches de cellules épithéliales. Nous avons démontré que des préparations enrichies en IgA provenant du plasma humain pouvaient diminuer les dommages et l'inflammation des monocouches induits par la toxine. L'ensemble de ces résultats démontrent que des IgA et IgM de type sécrétoire peuvent être générées à partir d'IgA et IgM issues du plasma humain en les associant à la pièce sécrétoire et que ces molécules protègent l'épithélium intestinal contre des bactéries pathogènes. Ces molécules pourraient dès lors être testées dans des modèles in vivo. Le but final serait de les utiliser chez l'homme à des fins d'immunisation passive dans le traitement de pathologies associées à la muqueuse telles que les infections. - Mucosal surfaces, such as gastrointestinal mucosa, are constantly exposed to the external environment and their protection is ensured by a combination of mechanical, physicochemical and immunological barriers. Among the various immunological defense mechanisms, specific humoral mucosal response plays a crucial role and is mediated by secretory immunoglobulins A (SIgA) and M (SIgM). Immunoglobulin therapy based on the administration of IgG molecules leads important health benefits. Even though animal studies have shown that mucosal application of polymeric IgA (plgA) or SIgA provided protection against infections, IgA/SIgA have been only used occasionally for therapeutic application. Moreover, preclinical and clinical studies have demonstrated that systemic administration of IgM-enriched preparations could also afford protection against infections. Nevertheless, mucosal application of purified IgM/SIgM has not been examined. The main reason is that the purification or production of IgA/SIgA and IgM/SIgM at large scale is difficult to achieve. The aim of this PhD project was to examine the possibility to associate polyclonal human plasma-derived IgA and IgM with recombinant human secretory component (SC) to generate functional secretoiy-like IgA and IgM. First, biochemical analysis of human plasma IgA and IgM was performed by western blotting and chromatography. These molecules exhibited the same biochemical features as mucosa-derived antibodies (Abs). The association between human plasma plgA or IgM and recombinant human SC was confirmed, as well as the 1:1 stoichiometry of association. Similarly to physiological conditions, this association delayed the degradation of secretory-like IgA or IgM by intestinal proteases. Secondly, the function activity and the mode of action of human plasma IgA and IgM, as well as secretory-like IgA and IgM were explored using an in vitro model of polarized intestinal epithelial Caco-2 cell monolayers mimicking intestinal epithelium. Cell monolayers were infected with an enteropathogen, Shigella flexneri, alone or in combination to plasma Abs or secretory-like Abs. Even though plasma plgA and secretoiy-like IgA resulted in a delay of bacteria-induced damages of cell monolayers, plasma IgM and secretory-like IgM were shown to be superior in maintenance of cell integrity. Polymeric IgA and secretory-like IgA induced bacterial aggregation and decreased cell monolayer inflammation, effects further amplified with IgM and secretory-like IgM. In addition, both secretory-like Abs directly impacted on bacterial virulence leading to a reduction in secretion of virulence factors by bacteria. The functionality of human plasma IgA was also tested against Clostridium difficile toxin A using Caco-2 cell monolayers. Human plasma IgA- enriched preparations led to a diminution of cell monolayer damages and a decrease of cellular inflammation induced by the toxin. The sum of these results demonstrates that secretory-like IgA and IgM can be generated from purified human plasma IgA and IgM associated to SC and that these molecules are functional to protect intestinal epithelium from bacterial infections. These molecules could be now tested using in vivo models. The final goal would be to use them by passive immunization in the treatment of mucosa-associated pathologies like infections in humans.

Transepithelial transport of HIV-1 by M cells is receptor-mediated.

Human colon carcinoma Caco-2 cell monolayers undergo conversion into cells that share morphological and functional features of M cells when allowed to interact with B lymphocytes. A lymphotropic (X4) HIV-1 strain crosses M cell monolayers and infects underlying CD4(+) target cells. Transport requires both lactosyl cerebroside and CXCR4 receptors, which are expressed on the apical surface of Caco-2 and M cells. Antibodies specific for each receptor block transport. In contrast, a monotropic (R5) HIV-1 strain is unable to cross M cell monolayers and infect underlying monocytes, despite efficient transport of latex beads. Caco-2 and M cells do not express CCR5, but transfection of these cells with CCR5 cDNA restores transport of R5 virus, which demonstrates that HIV-1 transport across M cells is receptor-mediated. The follicle-associated epithelium covering human gut lymphoid follicles expresses CCR5, but not CXCR4, and lactosyl cerebroside, suggesting that HIV-1 infection may occur through M cells and enterocytes at these sites.

Rôle et régulation de la protéine kinase AMPK au niveau intestinal

réalisé en cotutelle avec l'Université Claude Bernard Lyon 1

Anti-cancer properties of phenolics from apple waste on colon carcinogenesis in vitro

Colorectal cancer is one of the most common cancers in Western countries. The World Health Organisation identifies diet as a critical risk factor in the development and progression of this disease and the protective role of high levels of fruit and vegetable consumption. Several studies have shown that apples contain several phenolic compounds that are potent anti-oxidants in humans. However, little is known about other beneficial properties of apple phenolics in cancer. We have used the HT29, HT115 and CaCo-2 cell lines as in vitro models to examine the effect of apple phenolics (0.01–0.1% apple extract) on key stages of colorectal carcinogenesis, namely; DNA damage (Comet assay), colonic barrier function (TER assay), cell cycle progression (DNA content assay) and invasion (Matrigel assay). Our results indicate that a crude extract of apple phenolics can protect against DNA damage, improve barrier function and inhibit invasion (p < 0.05). The anti-invasive effects of the extract were enhanced with twenty-four hour pretreatment of cells (p < 0.05). We have shown that a crude apple extract from waste, rich in phenolic compounds, beneficially influences key stages of carcinogenesis in colon cells in vitro.

The fate of olive oil polyphenols in the gastrointestinal tract: implications of gastric and colonic microflora-dependent biotransformation

We have conducted a detailed investigation into the absorption, metabolism and microflora-dependent transformation of hydroxytyrosol ( HT), tyrosol (TYR) and their conjugated forms, such as oleuropein (OL). Conjugated forms underwent rapid hydrolysis under gastric conditions, resulting in significant increases in the amount of free HT and TYR entering the small intestine. Both HT and TYR transferred across human Caco-2 cell monolayers and rat segments of jejunum and ileum and were subject to classic phase I/II biotransformation. The major metabolites identified were an O-methylated derivative of HT, glucuronides of HT and TYR and a novel glutathionylated conjugate of HT. In contrast, there was no absorption of OL in either model. However, OL was rapidly degraded by the colonic microflora resulting in the formation of HT. Our study provides additional information regarding the breakdown of complex olive oil polyphenols in the GI tract, in particular the stomach and the large intestine.

A comparison of the anticancer properties of isoxanthohumol and 8-prenylnaringenin using in vitro models of colon cancer

The hops plant (Humulus lupulus L.) is an essential ingredient in beer and contains a number of potentially bioactive prenylflavonoids, the predominant being the weakly estrogenic isoxanthohumol (Ix), which can be converted to the more strongly estrogenic 8-PN by the colonic microbiota. The aim of this study was to investigate the biological activity of 8-PN and Ix using in vitro models representing key stages of colorectal carcinogenesis, namely cell growth and viability (MTT assay), cell-cycle progression (DNA content assay), DNA damage (Comet assay), and invasion (Matrigel assay). A significant decrease in Caco-2 cell viability was noted after both 8-PN and Ix treatments at the higher doses (40 and 50 μM, respectively) although the impact on cell cycle differed between the two compounds. The decreased cell viability observed after Ix treatment was associated with a concentration-dependent increase in G2/M and an increased sub-G1 cell-cycle fraction, whereas treatment with 8-PN was associated with an elevated G0/G1 and an increased sub-G1 cell-cycle fraction. Significant antigenotoxic activity was noted at all 8-PN concentrations tested (5-40 μM). Although significant antigenotoxic activity was also noted with Ix treatment at ≤25 μM, at a higher dose, Ix itself exerted genotoxic activity. In a dose-dependent manner, both compounds inhibited HT115 cell invasion with reductions up to 52 and 46% for Ix and 8-PN, respectively, in comparison to untreated cells. This study demonstrated that both Ix and its gut microbial metabolite 8-PN exert anticancer effects on models of key stages of colon tumourigenesis.

An exploratory study into the putative prebiotic activity of fructans isolated from Agave angustifolia and the associated anticancer activity

Linear Inulin type fructan (ITF) prebiotics have a putative role in the prevention of colorectal cancer, whereas relatively little is known about branched fructans. This study aims to investigate the fermentation properties and potential prebiotic activity of branched fructans derived from Agave angustifolia Haw, using the Simulator of Human Intestinal Microbial Ecosystem (SHIME) model. The proximal, transverse and distal vessels were used to investigate fructan fermentation throughout the colon and to assess the alterations of the microbial composition and fermentation metabolites (short chain fatty acids and ammonia). The influence on bioactivity of the fermentation supernatant was assessed by MTT, Comet and transepithelial electrical resistance (TER), respectively. Addition of Agave fructan to the SHIME model significantly increased (P<0.05), bifidobacteria populations (proximal and transverse), SCFA concentrations (proximal, transverse and distal) and decreased ammonia concentrations in the distal vessel. Furthermore, the fermentation supernatant significantly (P<0.05) increased the TER of a Caco-2 cell monolayer (%) and decreased fluorescein-based paracellular flux, suggesting enhanced barrier function and reduced epithelial barrier permeability (proximal and distal vessel). While cytotoxicity and genotoxicity remained unaltered in response to the presence of Agave fructans. To conclude, branched Agave fructans show indications of prebiotic activity, particularly in relation to colon health by exerting a positive influence on gut barrier function, an important aspect of colon carcinogenesis.

Avaliação toxicogenética e atividade antitumoral in vitro de complexos heterolépticos de Rutênio(II): Atividades citotóxicas, genotóxicas e interação com biomoléculas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Impact of folate absorption and transport for nutrition and drug targeting

In this thesis, interactions of folic acid with tea and tea components at the level of intestinal absorption have been investigated. Firstly, the interaction between folic acid and tea as well as tea catechins was studied in vitro, using Caco-2 cell monolayers and secondly, a clinical trial was designed and carried out. In addition, targeting of folic acid conjugated nanoparticles to FR expressing Caco-2 cells was studied in order to evaluate the principle of nutrient-receptor-coupled transport for drug targeting. In the first part of this work, it was shown that EGCG and ECG (gallated catechins) inhibit folic acid uptake (IC50 of 34.8 and 30.8 µmol/L) comparable to MTX (methotrexate) under these experimental conditions. Moreover, commercial green and black tea extracts inhibited folic acid uptake with IC50 values of approximately 7.5 and 3.6 mg/mL, respectively. These results clearly indicate an interaction between folic acid and green tea catechins at the level of intestinal uptake. The mechanism responsible for the inhibition process might be the inhibition of the influx transport routes for folates such as via RFC and/or PCFT. For understanding the in vivo relevance of this in vitro interaction, a phase one, open-labeled, randomized, cross-over clinical study in seven healthy volunteers was designed. For the 0.4 mg folic acid dose, the mean Cmax decreased by 39.2% and 38.6% and the mean AUC0 decreased by 26.6% and 17.9% by green tea and black tea, respectively. For the 5 mg folic acid dose, the mean Cmax decreased by 27.4% and mean AUC0 decreased by 39.9% when taken with green tea. The results of the clinical study confirm the interaction between tea and folic acid in vivo leading to lower bioavailabilities of folic acid. In the second part of the thesis, targeting studies using folic acid conjugated nanoparticles were conducted. Folic acid conjugated nanoparticles were shown to be internalized by the cell via FR (folate receptor) mediated endocytosis. DNA block copolymer micelles equipped with 2, 11 and 28 folic acid units respectively were applied on FR expressing Caco-2 cells. There was a direct proportion in the amount of internalized nanoparticle and the number of folic acid units on the periphery of the nanoparticle. To sum up, throughout this thesis, the importance of folic acid for nutrition and nutrient and drug related interactions of folic acid at intestinal level was shown. Furthermore, significance of FRs in targeting for cancer chemotherapy was demonstrated in in vitro cell culture experiments. Folic acid conjugated DNA block copolymer micelles were suggested as efficient nanoparticles for targeted drug delivery.

NHERF1 – New Modifier of Colorectal Cancer Progression

Colorectal cancer (CRC) develops from multiple progressive modifications of normal intestinal epithelium into adenocarcinoma. Loss of cell polarity has been implicated as an early event in this process, but the molecular players involved are not well known. NHERF1 (Na+/H+ Exchanger Regulatory Factor 1) is an adaptor protein with apical membrane localization in polarized epithelia. In this study, we tested our hypothesis that NHERF1 plays a role in CRC. We examined surgical CRC resection specimens for changes in NHERF1 expression, and modeled these changes in two- and three-dimensional (2D and 3D) Caco-2 CRC cell systems. NHERF1 had significant alterations from normal to adenoma and carcinoma transitions (2=38.5, d.f.=4, P<0.001), displaying apical membrane localization in normal tissue but loss of expression in adenoma and ectopic overexpression in carcinoma. In Caco-2 cell models, NHERF1 depletion induced epithelial-mesenchymal-transition in 2D cell monolayers and disruption of apical-basal polarity in 3D cyst system. The mesenchymal phenotype of NHERF1-depleted cells was fully restored by re-expression of NHERF1 at the apical membrane. Cytoplasmic and nuclear NHERF1 re-expression not only failed to restore the epithelial phenotype but led to more aggressive phenotypes. Our findings suggest that membrane NHERF1 is an important regulator of epithelial morphogenesis, and that changes in NHERF1 expression correlate with CRC progression. NHERF1 loss and ectopic expression that induce massive disruption of epithelial cell polarity may, thereby, mark important steps in CRC development.

IL-9 and its receptor are predominantly involved in the pathogenesis of UC.

OBJECTIVE Several pathogenic roles attributed over the past two decades to either T helper (Th)1 or Th2 cells are increasingly becoming associated with interleukin (IL)-17 and most recently IL-9 signalling. However, the implication of IL-9 in IBD has not been addressed so far. DESIGN We investigated the expression of IL-9 and IL-9R by using peripheral blood, biopsies and surgical samples. We addressed the functional role of IL-9 signalling by analysis of downstream effector proteins. Using Caco-2 cell monolayers we followed the effect of IL-9 on wound healing. RESULTS IL-9 mRNA expression was significantly increased in inflamed samples from patients with UC as compared with controls. CD3(+) T cells were major IL-9-expressing cells and some polymorphonuclear leucocytes (PMN) also expressed IL-9. IL-9 was co-localised with the key Th9 transcription factors interferon regulatory factor 4 and PU.1. Systemically, IL-9 was abundantly produced by activated peripheral blood lymphocytes, whereas its receptor was overexpressed on gut resident and circulating PMN. IL-9 stimulation of the latter induced IL-8 production in a dose-dependent manner and rendered PMN resistant to apoptosis suggesting a functional role for IL-9R signalling in the propagation of gut inflammation. Furthermore, IL-9R was overexpressed on gut epithelial cells and IL-9 induced STAT5 activation in these cells. Moreover, IL-9 inhibited the growth of Caco-2 epithelial cell monolayers in wound healing experiments. CONCLUSIONS Our results provide evidence that IL-9 is predominantly involved in the pathogenesis of UC suggesting that targeting IL-9 might become a therapeutic option for patients with UC.

REGULATION OF TOXIN SYNTHESIS BY CLOSTRIDIUM DIFFICILE

Clostridium difficile is the leading definable cause of nosocomial diarrhea worldwide due to its virulence, multi-drug resistance, spore-forming ability, and environmental persistence. The incidence of C. difficile infection (CDI) has been increasing exponentially in the last decade. Virulent strains of C. difficile produce either toxin A and/or toxin B, which are essential for the pathogenesis of this bacterium. Current methods for diagnosing CDI are mostly qualitative tests that detect the bacterium, the toxins, or the toxin genes. These methods do not differentiate virulent C. difficile strains that produce active toxins from non-virulent strains that do not produce toxins or produce inactive toxins. Based on the knowledge that C. difficile toxins A and B cleave a substrate that is stereochemically similar to the native substrate of the toxins, uridine diphosphoglucose, a quantitative, cost-efficient assay, the Cdifftox activity assay, was developed to measure C. difficile toxin activity. The concept behind the activity assay was modified to develop a novel, rapid, sensitive, and specific assay for C. difficile toxins in the form of a selective and differential agar plate culture medium, the Cdifftox Plate assay (CDPA). This assay combines in a single step the specific identification of C. difficile strains and the detection of active toxin(s). The CDPA was determined to be extremely accurate (99.8% effective) at detecting toxin-producing strains based on the analysis of 528 C. difficile isolates selected from 50 tissue culture cytotoxicity assay-positive clinical stool samples. This new assay advances and improves the culture methodology in that only C. difficile strains will grow on this medium and virulent strains producing active toxins can be differentiated from non-virulent strains. This new method reduces the time and effort required to isolate and confirm toxin-producing C. difficile strains and provides a clinical isolate for antibiotic susceptibility testing and strain typing. The Cdifftox activity assay was used to screen for inhibitors of toxin activity. Physiological levels of the common human conjugated bile salt, taurocholate, was found to inhibit toxin A and B in vitro activities. When co-incubated ex vivo with purified toxin B, taurocholate protected Caco-2 colonic epithelial cells from the damaging effects of the toxin. Furthermore, using a caspase-3 detection assay, taurocholate reduced the extent of toxin B-induced Caco-2 cell apoptosis. These results suggest that bile salts can be effective in protecting the gut epithelium from C. difficile toxin damage, thus, the delivery of physiologic amounts of taurocholate to the colon, where it is normally in low concentration, could be useful in CDI treatment. These findings may help to explain why bile rich small intestine is spared damage in CDI, while the bile salt poor colon is vulnerable in CDI. Toxin synthesis in C. difficile occurs during the stationary phase, but little is known about the regulation of these toxins. It was hypothesized that C. difficile toxin synthesis is regulated by a quorum sensing mechanism. Two lines of evidence supported this hypothesis. First, a small (KDa), diffusible, heat-stable toxin-inducing activity accumulates in the medium of high-density C. difficile cells. This conditioned medium when incubated with low-density log-phase cells causes them to produce toxin early (2-4 hrs instead of 12-16 hrs) and at elevated levels when compared with cells grown in fresh medium. These data suggested that C. difficile cells extracellularly release an inducing molecule during growth that is able to activate toxin synthesis prematurely and demonstrates for the first time that toxin synthesis in C. difficile is regulated by quorum signaling. Second, this toxin-inducing activity was partially purified from high-density stationary-phase culture supernatant fluid by HPLC and confirmed to induce early toxin synthesis, even in C. difficile virulent strains that over-produce the toxins. Mass spectrometry analysis of the purified toxin-inducing fraction from HPLC revealed a cyclic compound with a mass of 655.8 Da. It is anticipated that identification of this toxin-inducing compound will advance our understanding of the mechanism involved in the quorum-dependent regulation of C. difficile toxin synthesis. This finding should lead to the development of even more sensitive tests to diagnose CDI and may lead to the discovery of promising novel therapeutic targets that could be harnessed for the treatment C. difficile infections.

Comparison of selected methods for measuring the virulence properties of Listeria spp.

The comparative ability of different methods to assess virulence of Listeria species was investigated in ten Listeria strains. All strains were initially subjected to pulsed-field gel electrophoresis analysis to determine their relatedness. Virulence characteristics were subsequently tested for by (i) determining the presence of six virulence genes by polymerase chain reaction; (ii) testing for the production of listeriolysin O, phosphatidylcholine phospholipase C, and phosphatidylinositol-specific phospholipase C; (iii) investigating the hydrophobicity of the strains; (iv) determining the strains ability to attach to, enter, and replicate within the Caco-2 cells. Variations in most of the virulence characteristics were obvious across the strains for the range of tests performed. A wide range of anomalous results among methods were apparent. In particular, the presence of virulence genes was found to be unrelated to the production of virulence-associated proteins in vitro, while virulence protein production and hydrophobicity in Listeria monocytogenes were found to be unrelated or marginally related, respectively, to the ability to invade the Caco-2 cell line. It was concluded that the methods investigated were unable to consistently and unequivocally measure the differences in the virulence properties of the strains.

Iron (Fe) bioavailability and the distribution of anti-Fe nutrition biochemicals in the unpolished, and bran fraction of five rice gei polished grain riotypes

Iron (Fe) bioavailability in unpolished, polished grain and bran fraction of five rice genotypes with a range of Fe contents was measured by in vitro digestion and cultured Caco-2 cells of cooked grain. There was a significant difference in Fe bioavailability among the five rice genotypes tested, in both the unpolished and polished grain. The range of Fe bioavailability variation in polished rice was much wider than that of unpolished, suggesting the importance of using Fe levels and bioavailability in polished rice grain as the basis for selecting high-Fe rice cultivars for both agronomic and breeding purposes. Milling and polishing the grain to produce polished (or white) rice increased Fe bioavailability in all genotypes. Iron bioavailability in polished rice was high in the UBON2 and Nishiki, intermediate in both IR68144 and KDML105, and low in CMU122. All genotypes had low bioavailability of Fe in bran fraction compared to unpolished and polished grain, except in CMU122. CMU122 contained the lowest level of bioavailable Fe in unpolished and polished grain and bran, because of the dark purple pericarp colored grain and associated tannin content. The level of bioavailable Fe was not significantly correlated with grain Fe concentration or grain phytate levels among these five genotypes tested. The negative relationship between Fe bioavailability and the levels of total extractable phenol was only observed in unpolished (r = -0.83**) and bran fraction (r = -0.50*). The present results suggested that total extractable phenol and tannin contents could also contribute to lowering bioavailability of Fe in rice grain, in addition to phytate. (c) 2006 Society of Chemical Industry