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Many plant species within the terrestrial ecological zones of Canada have not yet been investigated for anti-cancer activity. We examined the scientific literature describing the endemic flora from the prairie ecological zone and selected the species, Thermopsis rhombifolia, locally known as the buffalo bean, for investigation of its anti-cancer potential. We tested it in cell-based assays using phenotypic screens that feature some of the hallmarks of cancer. An ethanolic extract prepared from T. rhombifolia was cytotoxic to HT-29 (colon) and SH-SY5Y (brain) cancer cell lines, and showed little cytotoxicity to a normal human cell line (WI-38). In phenotypic assays, we identified activities in the extracts that target cell death, cell cycle and cell adhesion. These data highlight the anti-cancer potential of previously untested plants found in northern ecological zones and the feasibility of using pertinent phenotypic assays to examine the anti-cancer potential of natural product extracts.

In the present paper, we report that mitosis is a key step in the cellular response to genotoxic agents in human cells. Cells with damaged DNA recruit γH2AX (phosphorylated histone H2AX), phosphorylate Chk1 (checkpoint kinase 1) and arrest in the G2-phase of the cell cycle. Strikingly, nearly all cells escape the DNA damage checkpoint and become rounded, by a mechanism that correlates with Chk1 dephosphorylation. The rounded cells are alive and in mitosis as measured by low phospho-Tyr15 Cdk1 (cyclin-dependent kinase 1), high Cdk activity, active Plk1 (Polo-like kinase 1) and high phospho-histone H3 signals. This phenomenon is independent of the type of DNA damage, but is dependent on pharmacologically relevant doses of genotoxicity. Entry into mitosis is likely to be caused by checkpoint adaptation, and the HT-29 cell-based model provides a powerful experimental system in which to explore its molecular basis. We propose that mitosis with damaged DNA is a biologically significant event because it may cause genomic rearrangement in cells that survive genotoxic damage.

Pathogens and eucaryotic cells are active partners during the process of pathogenicity. To gain access to enterocytes and to cross the epithelial membrane, many enterovirulent microorganisms interact with the brush border membrane-associated components as receptors. Recent reports provide evidence that intestinal cell differentiation plays a role in microbial pathogenesis. Human enteropathogenic Escherichia coli (EPEC) develop their pathogenicity upon infecting enterocytes. To determine if intestinal epithelial cell differentiation influences EPEC pathogenicity, we examined the infection of human intestinal epithelial cells by JPN 15 (pMAR7) [EAF+ eae+] EPEC strain as a function of the cell differentiation. The human embryonic intestinal INT407 cells, the human colonic T84 cells, the human undifferentiated HT-29 cells (HT-29 Std) and two enterocytic cell lines, HT-29 glc−/+ and Caco-2 cells, were used as cellular models. Cells were infected apically with the EPEC strain and the cell-association and cell-entry were examined by quantitative determination using metabolically radiolabeled bacteria, as well as by light, scanning and tranmission electron microscopy. [EAF+ eae+] EPEC bacteria efficiently colonized the cultured human intestinal cells. Diffuse bacterial adhesion occurred to undifferentiated HT-29 Std and INT407 cells, whereas characteristic EPEC cell clusters were observed on fully differentiated enterocytic HT-29 glc−/+ cells and on colonic crypt T84 cells. As shown using the Caco-2 cell line, which spontaneously differentiates in culture, the formation of EPEC clusters increased as a function of the epithelial cell differentiation. In contrast, efficient cell-entry of [EAF+ eae+] EPEC bacteria occured in recently differentiated Caco-2 cells and decreased when the cells were fully differentiated. Our results provide evidence that the intestinal cell differentiation could play a dual role in EPEC pathogenesis: it up-regulates intestinal cell colonization and down-regulates intestinal cell invasion.

Gene silencing via heterochromatin formation plays a major role in cell differentiation and maintenance of homeostasis. Here we report the identification and characterization of a novel heterochromatinization factor in vertebrates, bromo adjacent homology domain–containing protein 1 (BAHD1). This nuclear protein interacts with HP1, MBD1, HDAC5, and several transcription factors. Through electron and immunofluorescence microscopy studies, we show that BAHD1 overexpression directs HP1 to specific nuclear sites and promotes the formation of large heterochromatic domains, which lack acetyl histone H4 and are enriched in H3 trimethylated at lysine 27 (H3K27me3). Furthermore, ectopically expressed BAHD1 colocalizes with the heterochromatic inactive X chromosome (Xi). The BAH domain is required for BAHD1 colocalization with H3K27me3, but not with the Xi chromosome. As highlighted by whole genome microarray analysis of BAHD1 knockdown cells, BAHD1 represses several proliferation and survival genes, in particular the insulin-like growth factor II gene ( IGF2 ). When overexpressed, BAHD1 specifically binds the CpG-rich P3 promoter of IGF2 , which increases MBD1 and HDAC5 targeting at this locus. This region contains DNA-binding sequences for the transcription factor SP1, with which BAHD1 coimmunoprecipitates. Collectively, these findings provide evidence that BAHD1 acts as a silencer by recruiting at specific promoters a set of proteins that coordinate heterochromatin assembly.

Leptospires exist as saprophytic organisms that are aquatic or as pathogens that are able to survive in water. Leptospirosis is transmitted to humans through environmental surface waters contaminated by the urine of mammals, usually rodents, which are chronically infected by pathogenic strains. The ecology of Leptospira spp. prompted us to evaluate if these spirochaetes were able to form biofilms. This study investigated the characteristics of biofilm development by both saprophytic and pathogenic Leptospira species using microscopic examinations and a polystyrene plate model. Biofilms were formed preferentially on glass and polystyrene surfaces. Electron microscopic images showed cells embedded in an extracellular matrix. The formation of such a biofilm is consistent with the life of saprophytic strains in water and may help pathogenic strains to survive in environmental habitats and to colonize the host.

Summary b 1 integrins are anchored on the basal membrane of enterocytes, but little is known about their localization in M cells, which are the main entry route into the intestinal mucosa for many bacterial pathogens. In particular, it has been suggested that adhesion of enteropathogenic Yersinia to M cells is mediated by interaction of the bacterial protein invasin and apical b 1 integrins. Using a novel in vitro model of M cells, we demonstrate an augmented apical and basolateral targeting of b 1 integrins in M cells associated with increased total a chain synthesis. The a a a 3 and a a a 6 subunits were targeted to the basal pole, but a 2 subunit was targeted at both poles. No other a a a subunit

In the intestine, the follicle-associated epithelium (FAE) of Peyer’s patches (PP) performs Ag sampling as the first step in developing immune responses. Depending on the species, this epithelium contains 10–50% of M cells, which act as regulated gates in epithelial barriers that can be used opportunistically by pathogens to invade their host. However, the mechanisms involved in the differentiation and uptake processes of M cells are not known, in part because their limited number in the intestinal mucosa has hampered molecular and biochemical studies. In this work we provide evidence that PP lymphocytes can themselves modulate gene expression in PP in vivo and in an in vitro model of FAE. Transgenic mice carrying a reporter gene under the control of a modified l-pyruvate kinase promoter (SVPK) exhibit strong transgene expression in PP and FAE, but not in the adjacent villous cells. We used the mouse intestinal epithelial cell line m-ICcl2 transfected with the SVPK promoter fused to β-galactosidase to investigate the direct effect of PP lymphocytes on SVPK promoter activity. β-Galactosidase expression was 4.4-fold higher in transfected m-ICcl2 cells when they were cultured with PP lymphocytes. Conversely, green fluorescent protein expression was 1.8-fold lower in stably transfected differentiated intestinal Caco-2cl1 cells with the sucrase isomaltase promoter fused to green fluorescent protein cDNA when they were cultured with PP lymphocytes, indicating that the in vivo FAE down-regulation of sucrase isomaltase promoter is transcriptionally regulated.

Ribosomal preparations of pathogenic micro-organisms of the upper respiratory tract can be delivered orally for the prevention of recurrent infectious episodes, because they induce mucosal and protective immune responses. The mechanism of mucosal barrier translocation is difficult to study in animal models, little is therefore known about this process. In order to circumvent these problems, we have examined the uptake of ribosomal preparations in three experimental systems that model human intestinal cells. We found that M-like cells displayed a 8.7-fold increase in the uptake of a ribosomal immunostimulant when compared to absorptive or crypt enterocyte-like cells. The product was taken up, translocated, and delivered in the basolateral compartment by cultured M-like cells. No translocation was observed across monolayers of T84 cells (model of crypt cells). Only minimal translocation occured through monolayers of Caco-2 cells (model of absorptive enterocytes). This suggests that, in vivo, colyophilisat is delivered mainly through the M cells overlying lymphoid follicles (Peyer's patches) or nodules of the gut-associated mucosal lymphoid tissue, which are the major inductor sites of mucosal responses. Use of the M-like cell cultured model could be a key step for the development of even more efficient immunostimulators in animals and human.

Peyer's patch lymphocytes cocultured with Caco-2 cells trigger the phenotypic conversion of enterocytes into cells that express morphological and functional M-cell properties. We report a technical update for setting up this model, which will enable the study of M-cell biology, the identification by biochemical approaches of molecules involved in the interaction of microorganisms with M cells, and the development of vectors that would efficiently target the mucosal immune system.

Yersinia enterocolitica cross the intestinal epithelium via translocation through M cells, which are located in the follicle-associated epithelium (FAE) of Peyer's patches (PP). To investigate the molecular basis of this process, studies were performed using a recently developed in vitro model, in which the enterocyte-like cell line Caco-2 and PP lymphocytes are co-cultured in order to establish FAE-like structures including M cells. Here, we demonstrate that Y. enterocolitica does not adhere significantly to the apical membrane of differentiated enterocyte-like Caco-2 cells that express binding sites for Ulex europaeus agglutinin (UEA)-1. In contrast, Y. enterocolitica adhered to, and was internalized by, cells that lacked UEA-1 binding sites and displayed a disorganized brush border. These cells were considered to be converted to M-like cells. Further analysis revealed that part of these cells expressed beta1 integrins at their apical surface and, as revealed by comparison of wild-type and mutant strains, interacted with invasin of Y. enterocolitica. Consistently, anti-beta1 integrin antibodies significantly inhibited internalization of inv-expressing yersiniae. Experiments with Yersinia mutant strains deficient in YadA or Yop secretion revealed that these virulence factors play a minor role in this process. After internalization, yersiniae were transported within LAMP-1-negative vacuoles to, and released at, the basal surface. Internalization and transport of yersiniae was inhibited by cytochalasin D, suggesting that F-actin assembly is required for this process. These results provide direct evidence that expression of beta1 integrins at the apical surface of M cells enables interaction with the invasin of Y. enterocolitica, and thereby initiates internalization and translocation of bacteria.

The epithelium that lines the gut is impermeable to macromolecules and microorganisms, except in Peyer's patches (PPs), where the lymphoid follicle-associated epithelium (FAE) contains M cells that transport antigens and microorganisms. A cultured system that reproduces the main characteristics of FAE and M cells was established by cultivation of PP lymphocytes with the differentiated human intestinal cell line Caco-2. Lymphocytes settled into the epithelial monolayer, inducing reorganization of the brush border and a temperature-dependent transport of particles and Vibrio cholerae . This model system could prove useful for intestinal physiology, vaccine research, and drug delivery studies.

The association of enterotoxigenic Escherichia coli expressing colonization factor antigen I (CFA/I) with the cultured human colon adenocarcinoma cell, a model of the mature enterocyte of the small intestine, is dependent on the binding of CFA/I to a brush border-associated component. Binding of the purified radiolabeled [125I]CFA/I- and 14C-labeled CFA/I-positive bacteria could be displaced by an increasing concentration of unlabeled CFA/I. Moreover, we showed that expression of the specific CFA/I binding developed as a function of cell differentiation in Caco-2 cells, whereas expression of the nonspecific binding did not. Expression of the brush border differentiation-associated component acting as a binding site for CFA/I was up-regulated by glucose. Indeed, the enterocyte-like HT-29 glc- cell subpopulation not expressing the CFA/I binding site when cultured in dialyzed serum and hexose-free medium regained the ability to bind CFA/I when the cells were returned to culture medium containing glucose. Furthermore, expression of the brush border-associated CFA/I binding site in the enterocyte-like Caco-2 cells was repressed when the cells were cultured in hexose-free conditions.

BACKGROUND & AIMS: The follicle-associated epithelium (FAE) of Peyer's patches mainly consists of two cell types: absorptive enterocytes with a brush border and M cells without this apical specialization. To study the controversial ontogeny of M cells (mesenchymal vs. epithelial origin), the expression pattern of tissue-specific cytoskeletal proteins, markers of cell origin that play a crucial role in the specific shape of epithelial cells and brush border assembly, was investigated. METHODS: The localization of cytokeratins, vimentin, and villin was determined on mouse FAE using immunocytochemistry and electron microscopy. RESULTS: Epithelial-specific cytokeratins were expressed in both absorptive enterocytes and M cells, whereas vimentin was not detected in mouse FAE. Villin, a tissue-specific, actin-binding protein of the brush border, was expressed in the two cell types. This protein had an unusual cytoplasmic distribution in FAE cells lacking a brush border and in cells having an intraepithelial pocket filled with lymphocytes. CONCLUSIONS: The presence of villin and the absence of vimentin in M cells support the intestinal origin of M cells. The cytoplasmic distribution of villin provides a new identification criteria for M cells and reflects the reorganization of the F-actin network, which correlates with the inability of M cells to assemble a brush border. (Gastroenterology 1996 Feb;110(2):515-21)

Enterotoxigenic Escherichia coli (ETEC) bearing the fimbrial colonisation factor antigens CFA/I, CFA/II, CFA/III, and the non-fimbrial antigen 2230 were tested for their ability to adhere to two cultured human intestinal HT-29 mucus secreting cell subpopulations. These populations are referred to as HT29-MTX and HT29-FU, which differ in the amount of secreted mucins and in their gastric or colonic mucin immunoreactivity respectively. Adherence of radiolabelled bacteria to cell monolayers infected apically was assessed. All ETEC strains adhered to the mucus secreting HT29-FU subpopulation, which secretes mucins of colonic immunoreactivity. Visualisation of bacteria by scanning electron microscopy showed that ETEC bound to the HT29-FU cells possessing a brush border, but not to the mucus and that ETEC binding developed as a function of cell differentiation. The adhesion of ETEC to cells possessing a brush border and to mucus secreting cells was also analysed by indirect immunofluorescence in HT29-MTX cells, which secrete mucins of gastric immunoreactivity. Fluorescein isothiocyanate labelling using specific anti-CFA/I antibody was used to show ETEC; rhodamine isothiocyanate labelling using a monoclonal antibody (designated M1) against purified human gastric mucus was used to detect secreted mucins, and rhodamine isothiocyanate labelling using a monoclonal antibody (designated 4H3) against human dipeptidylpeptidase IV was used to show cells possessing a brush border. Binding of bacteria colocalised with dipeptidylpeptidase IV of enterocytes and not with mucins.

We have recently demonstrated that cultured human intestinal HT-29 and Caco-2 cell lines express receptors for the F1845 fimbrial adhesin harbored by the diarrheagenic C1845 Escherichia coli (Kerneis et al., Infect. Immun. 59 (1991) 4013–4018). This adhesinn belongs to a family of adhesins including the Dr hemagglutinin and the afimbrial adhesin AFA-1 harbored by uropathogenic E. coli. Here we investigated the cell association of laboratory E. coli strains expressing the Dr hemagglutinin and the afimbrial adhesin AFA-I with human cultured enterocyte-like or mucosecreting cells. We observed that the E. coli strains bearing these adhesins adhere both to human intestinal undifferentiated and differentiated fluid-transporting cells, and to mucus-secreting cells. This result strongly suggests a high capacity of intestinal colonization for the uropathogenic E. coli harboring adhesive factors belonging to the Dr adhesin family. These results further corroborate the intestinal colonization by uropathogenic E. coli of the Dr family related to the fecal-perineal-urethral hypothesis of urinary tract infection pathogenesis.

Twenty-five strains of lactobacilli were tested for their ability to adhere to human enterocyte-like Caco-2 cells in culture. Seven Lactobacillus strains adhered well to the Caco-2 cells, of which three possessed calcium-independent adhesion properties. A high level of calcium-independent adhesion was observed with the human stool isolate Lactobacillus acidophilus strain LB. Scanning electron microscopy revealed that this strain adhered to the apical brush border of the cells. Adhesion increased in parallel with the morphological and functional differentiation of the Caco-2 cells. Two Lactobacillus components were involved in this adhesion. One was protease-resistant and bacterial-surface-associated; the other was heat-stable, extracellular and protease-sensitive.

Whole diffusely adhering Escherichia coli (DAEC) C1845 cells bearing the F1845 adhesive factor bind diffusely to differentiated human colon carcinoma cell lines HT-29 and Caco-2. By using antibodies directed against the purified fimbrial adhesin F1845 factor, the expression of the DAEC F1845-specific brush border receptors in the polarized human intestinal HT-29 and Caco-2 epithelial cells was studied by indirect immunofluorescence. A low level of DAEC F1845 receptors in undifferentiated intestinal cells was detected; they were localized in a cluster of cells. DAEC F1845 receptors were expressed at a high level in differentiated HT-29 and Caco-2 cells. DAEC F1845 receptors were expressed at a strikingly high level in the apical domains of the cells and developed during enterocytic differentiation in culture, in parallel with the apical expression of the intestinal brush border hydrolase, sucrase-isomaltase.

The current paradigm in Plasmodium falciparum malaria pathogenesis states that young, ring-infected erythrocytes (rings) circulate in peripheral blood and that mature stages are sequestered in the vasculature, avoiding clearance by the spleen. Through ex vivo perfusion of human spleens, we examined the interaction of this unique blood-filtering organ with P falciparum–infected erythrocytes. As predicted, mature stages were retained. However, more than 50% of rings were also retained and accumulated upstream from endothelial sinus wall slits of the open, slow red pulp microcirculation. Ten percent of rings were retained at each spleen passage, a rate matching the proportion of blood flowing through the slow circulatory compartment established in parallel using spleen contrast-enhanced ultrasonography in healthy volunteers. Rings displayed a mildly but significantly reduced elongation index, consistent with a retention process, due to their altered mechanical properties. This raises the new paradigm of a heterogeneous ring population, the less deformable subset being retained in the spleen, thereby reducing the parasite biomass that will sequester in vital organs, influencing the risk of severe complications, such as cerebral malaria or severe anemia. Cryptic ring retention uncovers a new role for the spleen in the control of parasite density, opening novel intervention opportunities.

Beta1 integrins are anchored on the basal membrane of enterocytes, but little is known about their localization in M cells, which are the main entry route into the intestinal mucosa for many bacterial pathogens. In particular, it has been suggested that adhesion of enteropathogenic Yersinia to M cells is mediated by interaction of the bacterial protein invasin and apical beta1 integrins. Using a novel in vitro model of M cells, we demonstrate an augmented apical and basolateral targeting of beta1 integrins in M cells associated with increased total alpha chain synthesis. The alpha3 and alpha6 subunits were targeted to the basal pole, but alpha2 subunit was targeted at both poles. No other alpha subunit was found associated with apical beta1 integrins on M cells. Interestingly, Y. enterocolitica still adhered to the apical surface of M cells, despite the fact that alpha2beta1 is not a receptor for invasin. We therefore studied the adhesive properties of invasin-mutant Y. enterocolitica and invasin-expressing Escherichia coli on the apical surface of M cells. We show that it is not invasin, but the product of an as yet unidentified bacterial chromosomal gene, that is involved in the adhesion of Y. enterocolitica to the apical membrane of M cells.

Rabbit appendix consists mainly of lymphoid follicles (LF) covered by M cells, the specialized antigen-sampling cells of the mucosal immune system, and surrounded by glandular epithelium. Until now, these M cells have been characterized morphologically and histologically by using cellular markers. Here, the adhesion and transport of pathogenic bacteria were investigated to assess the function of M cells of the appendix. We used the enteroinvasive motile Salmonella typhimurium and the rabbit enteropathogenic non-motile Escherichia coli RDEC-1, which are known to target specifically rabbit M cells of Peyer's patches (PPs). We found that S. typhimurium efficiently attached and was transported through appendix M cells in vivo. In contrast to S. typhimurium, RDEC-1 targeted M cells only ex vivo, when bacteria were allowed to have direct contact with the surface of the follicle. The difference in interaction of the two bacteria with appendix M cells led us to investigate whether this could be correlated with the lack of motility of RDEC-1. We used an aflagellate mutant of S. typhimurium and found that it had the same infection phenotype as RDEC-1. Gene complementation restored the efficiency of infection to that of S. typhimurium wild-type strain. In conclusion, we show that M cells of the appendix display features of the canonical M cells of PP, since they efficiently sample luminal pathogenic bacteria. However, due to the morphology of the appendix, motile bacteria appear to be more potent in their interactions with appendix M cells.

The maintenance during adult life of a large spectrum of pluripotency by stem cells originating from the endoderm seems to be the grounds for the striking plasticity of the digestive epithelium, which is able to drastically modify its differentiation pattern depending on the microenvironment. As a paradigm, Peyer's patch M cell development appears to be induced by crosstalk between lymphoid cells and/or microorganisms. Examples of pathological transdifferentiation of epithelia, also described as 'metaplasia' and affecting various organs, support the concept of intestinal plasticity. Though, the molecular processes involved in epithelial transdifferentiation have not been identified, histological analyses of these metaplastic tissues and experimental induction of transdifferentiation of normal epithelia provide lines of evidence suggesting that a modification of the local environment, such as occurs during contact of the epithelium with lymphoid cells or microorganisms, plays a key role in this process.

Salmonella typhimurium and enteropathogenic Escherichia coli (EPEC) were found to adhere to the brush border of differentiated human intestinal epithelial Caco-2 cells in culture, whereas Yersinia pseudotuberculosis and Listeria monocytogenes adhered to the periphery of undifferentiated Caco-2 cells. All these enterovirulent strains invaded the Caco-2 cells. Using a heat-killed human Lactobacillus acidophilus (strain LB) which strongly adheres both to undifferentiated and differentiated Caco-2 cells, we have studied inhibition of cell association with and invasion within Caco-2 cells by enterovirulent bacteria. Living and heat-killed Lactobacillus acidophilus strain LB inhibited both cell association and invasion of Caco-2 cells by enterovirulent bacteria in a concentration-dependent manner. The mechanism of inhibition of both adhesion and invasion appears to be due to steric hindrance of human enterocytic pathogen receptors by whole-cell lactobacilli rather than to a specific blockade of receptors.

Summary Experiments reported in this communication showed that the highly toxinogenic Cd 79685, Cd 4784, and Wilkins Clostridium difficile strains and the moderately toxinogenic FD strain grown in the presence of blood adhere to polarized monolayers of two cultured human intestinal cell lines: the human colonic epithelial Caco-2 cells and the human mucus-secreting HT29-MTX cells. Scanning electron microscopy revealed that the bacteria interacted with well-defined apical microvilli of differentiated Caco-2 cells and that the bacteria strongly bind to the mucus layer that entirely covers the surface of the HT29-MTX cells. The binding of C. difficile to Caco-2 cells developed in parallel with the differentiation features of the Caco-2 cells, suggesting that the protein(s) which constitute C. difficile-binding sites are differentiation-related brush border protein(s). To better define this interaction, we tentatively characterized the mechanism(s) of adhesion of C. difficile with adherence assays. It was shown that heating of C. difficile grown in the presence of blood enhanced the bacterial interaction with the brush border of the enterocyte-like Caco-2 cells and the human mucus-secreting HT29-MTX cells. A labile surface-associated component was involved in C. difficile adhesion since washes of C. difficile grown in the presence of blood without heat shock decreased adhesion. After heating, washes of C. difficile grown in the presence of blood did not modify adhesion. Analysis of surface-associated proteins of C. difficile subjected to different culture conditions was con-ducted. After growth of C. difficile Cd 79685, Cd 4784, FD and Wilkins strains in the presence of blood and heating, two predominant SDS-extractable proteins with molecular masses of 12 and 27 kDa were observed and two other proteins with masses of 48 and 31 kDa disappeared. Direct involvement of the 12 and 27 kDa surface-associated proteins in the adhe-sion of C. difficile strains was demonstrated by using rat polycolonal antibodies pAb 12 and pAb 27 directed against the 12 and 27kDa proteins. Indeed, adhesion to Caco-2 cell monoiayers of C. difficiie strains grown in the presence of blood, without or with heat-shock, was blocked. Taken together, our results suggest that C. difficiie may utilize blood components as adhesins to adhere to human intestinal cultured cells.

To study the expression of human intestinal receptors for enterotoxigenic Escherichia coli (ETEC), the human polarized intestinal epithelial cell line Caco-2 in culture and several subpopulations of HT-29 cells in culture--parental (mainly undifferentiated) HT-29 cells (HT-29 Std), an enterocytelike subpopulation obtained by selection through glucose deprivation (HT-29 Glc-), and an enterocytelike subpopulation obtained by selection through glucose deprivation which maintains its differentiation characteristics when switched back to standard glucose-containing medium (HT-29 Glc-/+)--were used. Since Caco-2 spontaneously differentiated in culture under standard culture conditions (in the presence of glucose) and HT-29 cells were undifferentiated when cultured under standard conditions (HT-29 Std) and differentiated when grown in a glucose-free medium (HT-29 Glc-), we studied the expression of the receptors for colonization factor antigens (CFA) I, II, and III and the 2230 antigen of ETEC in relation to enterocytic differentiation. We provide evidence that expression of ETEC CFA receptors develops in parallel with other differentiation functions of the cultured cells. The expression of ETEC-specific brush border receptors was studied by indirect immunofluorescence using antibodies raised against purified ETEC CFA. No ETEC receptors were detected in HT-29 Std or short-term-cultured Caco-2 cells. However, among the population of HT-29 Std cells, 2 to 4% of the cells were found to bind ETEC, and these cells expressed positive carcinoembryonic antigen immunoreactivity. This indicated that among the population of undifferentiated HT-29 cells, clusters of differentiated cells were present. ETEC CFA receptors were expressed in the apical and basolateral domains of differentiated HT-29 cells, whereas in differentiated Caco-2 cells only apical expression was observed. Both in HT-29 cells (HT-29 Glc-/+) and in Caco-2 cells cultured under standard conditions, ETEC CFA receptors develop as a function of day in culture. This indicated that the expression of the ETEC CFA receptors was a growth-related event. Indeed, ETEC CFA receptors developed in step with the apical expression of differentiation-associated proteins.

The adhesion of Lactobacillus acidophilus BG2FO4, a human stool isolate, to two human enterocytelike cell lines (Caco-2 and HT-29) and to the mucus secreted by a subpopulation of mucus-secreting HT29-MTX cells was investigated. Scanning electron microscopy revealed that the bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage and with the mucus secreted by the subpopulation of HT29-MTX cells. The adhesion to Caco-2 cells did not require calcium and involved an adhesion-promoting factor that was present in the spent supernatant of L. acidophilus cultures. This factor promoted adhesion of poorly adhering human Lactobacillus casei GG but did not promote adhesion of L. casei CNRZ 387, a strain of dairy origin. The adherence components on the bacterial cells and in the spent supernatant were partially characterized. Carbohydrates on the bacterial cell wall appeared to be partly responsible for the interaction between the bacteria and the extracellular adhesion-promoting factor. The adhesion-promoting factor was proteinaceous, since trypsin treatment dramatically decreased the adhesion of the L. acidophilus strain. The adhesion-promoting factor may be an important component of Lactobacillus species that colonize the gastrointestinal tract.

Diarrheagenic Escherichia coli (ETEC) bearing CFA/I or CFA/II adhesive factors specifically adhere onto the brush border of the polarized epithelial human intestinal Caco-2 cells in culture. Heat-killed Lactobacillus acidophilus strain LB, that adheres onto Caco-2 cells, inhibits diarrheagenic Escherichia coli adhesion in a concentration-dependent manner. Since the L. acidophilus does not express ETEC-CFA adhesive factors, it can be postulated that the heat-killed L. acidophilus LB cells inhibit diarrheagenic E. coli attachment by steric hindrance of the human enterocytic ETEC receptors.

Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion., Author Summary Entamoeba histolytica is the causative agent of amoebiasis, a human disease. Like other enteric infections, the lack of animal models enhances the difficulty of studying the development of amoebiasis. To date, no experimental model has been developed that reproduces the invasive intestinal amoebic lesions seen in human colon. We present the first study that examines, using human colon explants, the early steps of the human colonic barrier invasion by E. histolytica. With this ex vivo integrative model we have investigated both parasite behaviour and the human tissue response. Remarkably, in this model E. histolytica was able to cross and destroy the intestinal barrier evoking a tissue inflammatory response, while E. dispar, a non-pathogenic species, was unable to penetrate nor induce tissue responses. Furthermore, we have explored the role of three virulence factors during the invasive process, using gene-silenced E. histolytica trophozoites, particularly the kinetics of invasion, tissue destruction and induction of an early inflammatory responses. This is, to our knowledge, the first time that their role is highlighted in a complex human system. Our study provides new insights in the molecular mechanisms involved in the early steps of human colon invasion by E. histolytica.