Mucus-secreting HT29-MTX Cells Research Articles

Loss of Mucosal p32/gC1qR/HABP1 Triggers Energy Deficiency and Impairs Goblet Cell Differentiation in Ulcerative Colitis.

Background & AimsCell differentiation in the colonic crypt is driven by a metabolic switch from glycolysis to mitochondrial oxidation. Mitochondrial and goblet cell dysfunction have been attributed to the pathology of ulcerative colitis (UC). We hypothesized that p32/gC1qR/HABP1, which critically maintains oxidative phosphorylation, is involved in goblet cell differentiation and hence in the pathogenesis of UC.MethodsEx vivo, goblet cell differentiation in relation to p32 expression and mitochondrial function was studied in tissue biopsies from UC patients versus controls. Functional studies were performed in goblet cell-like HT29-MTX cells in vitro. Mitochondrial respiratory chain complex V-deficient, ATP8 mutant mice were utilized as a confirmatory model. Nutritional intervention studies were performed in C57BL/6 mice.ResultsIn UC patients in remission, colonic goblet cell differentiation was significantly decreased compared to controls in a p32-dependent manner. Plasma/serum L-lactate and colonic pAMPK level were increased, pointing at high glycolytic activity and energy deficiency. Consistently, p32 silencing in mucus-secreting HT29-MTX cells abolished butyrate-induced differentiation and induced a shift towards glycolysis. In ATP8 mutant mice, colonic p32 expression correlated with loss of differentiated goblet cells, resulting in a thinner mucus layer. Conversely, feeding mice an isocaloric glucose-free, high-protein diet increased mucosal energy supply that promoted colonic p32 level, goblet cell differentiation and mucus production.ConclusionWe here describe a new molecular mechanism linking mucosal energy deficiency in UC to impaired, p32-dependent goblet cell differentiation that may be therapeutically prevented by nutritional intervention.

Repeated exposure of Caco-2 versus Caco-2/HT29-MTX intestinal cell models to (nano)silver in vitro: Comparison of two commercially available colloidal silver products

Colloidal silver products are sold for a wide range of disinfectant and health applications. This has increased the potential for human exposure to silver nanoparticles (AgNPs) and ions (Ag+), for which oral ingestion is considered to be a major route of exposure. Our objective was to evaluate and compare the toxicity of two commercially available colloidal silver products on two human intestinal epithelial models under realistic exposure conditions.Mesosilver™ and AgC were characterized and a concentration range between 0.1 and 12 μg/mL chosen. Caco-2 cells vs. co-culture of Caco-2 and mucus-secreting HT29-MTX cells (90/10) were used. Repeated exposure was carried out to determine cell viability over 18 days of cell differentiation in 24-well plates. Selected concentrations (0.1, 1, and 3 μg/mL) were tested on cells cultured in E-plates and Transwells with the same repeated exposure regimen, to determine cell impedance, and cell viability and trans-epithelial electrical resistance (TEER), respectively. Silver uptake, intracellular localisation, and translocation were determined by CytoViva™, HIM-SIMS, and ICP-MS. Genotoxicity was determined on acutely-exposed proliferating Caco-2 cells by γH2AX immunofluorescence staining.Repeated exposure of a given concentration of AgC, which is composed solely of ionic silver, generally exerted more toxic effects on Caco-2 cells than Mesosilver™, which contains a mix of AgNPs and ionic silver. Due to its patchy structure, the presence of mucus in the Caco-2/HT29-MTX co-culture only slightly mitigated the deleterious effects on cell viability. Increased genotoxicity was observed for AgC on proliferating Caco-2 cells. Silver uptake, intracellular localisation, and translocation were similar.In conclusion, Mesosilver™ and AgC colloidal silver products show different levels of gut toxicity due to the forms of distinct silver (AgNPs and/or Ag+) contained within. This study highlights the applicability of high-resolution (chemical) imaging to detect and localize silver and provides insights into its uptake mechanisms, intracellular fate and cellular effects.

Probiotic Lactobacillus strains from Mongolia improve calcium transport and uptake by intestinal cells in vitro

The aim of this study was to investigate the probiotic properties of 174 Lactobacillus strains isolated from Mongolian dairy products, and particularly their impact on intestinal calcium uptake and absorption. All isolates underwent a first screening based on cell surface hydrophobicity, acid tolerance, tolerance to gastro-intestinal digestion, autoaggregation, adhesion and cytotoxicity against intestinal cells. Six Lactobacillus strains from different species (L. casei, L. kefiranofaciens, L. plantarum, L. fermentum, L. helveticus and L. delbrueckii) were selected, and their impact on intestinal calcium uptake and transport was investigated using Caco-2. Five strains were able to improve total calcium transport after 24 h contact with a differentiated Caco-2 cell monolayer. Concomitantly the L. plantarum strain was able to increase cellular calcium uptake in Caco-2 cells by 10% in comparison to control conditions. To determine which pathway(s) of calcium absorption was modulated by the strains, a qPCR-based study on 4 genes related to calcium/vitamin D metabolism or tight junction integrity was conducted on mucus-secreting intestinal HT-29 MTX cells. The L. plantarum strain modulates the transcellular pathway by regulating the expression of vitamin D receptor (1.79 fold of control) and calcium transporter (4.77 fold of control) while the L. delbrueckii strain acts on the paracellular pathway by modulating claudin-2 expression (2.83 fold of control). This work highlights the impact of Lactobacillus probiotic strains on intestinal calcium absorption and for the first time give some evidence about the cellular pathways involved.

P006 3D Multicellular intestine-on-a-chip model for disease modelling and drug discovery

Abstract Background One of the major functions of the human intestine is to provide a protective epithelial barrier between the body and digestive environment. Additionally, the interplay of commensal microbes of the gut microbiome with the gut tissue and host immune system significantly contributes to intestinal homeostasis. Crohn’s disease and ulcerative colitis, collectively referred to as inflammatory bowel diseases, are both associated with increased permeability of the epithelial barrier and dysregulated immune response. Great efforts have been made to develop both in vitro and in vivo models of the human intestine in order to understand the development and underlying pathogenesis of IBD. These efforts have provided valuable insights into multiple aspects of the disease. However, none of these models has been able to capture the complexity and multifactorial nature of IBD. Animal models generally fail to accurately predict the efficacy and toxicity of novel compounds in human tissues, while in vitro human intestinal models developed on porous membranes within Transwell inserts fail to accurately recapitulate and mimic key aspects of the in vivo situation. Methods Here, we present the development and characterisation of a 3D multicellular perfused intestine-on-a-chip model in a microfluidic platform, the OrganoPlate®, and its application for investigating intestinal inflammation. The model described here comprises a coculture of Caco-2 and mucus-secreting HT29-MTX cells in the top compartment of the chip and a coculture of immune-competent cells THP-1 and MUTZ-3 in the bottom compartment, lining a collagen-I ECM in the middle. Results We show that the Caco-2 and HT29-MTX coculture form confluent and polarised tubular structures against the collagen-I ECM in the OrganoPlate®, with a stable barrier function over time as well as the capability to secrete mucus. By exposing the cultures to TNFα and/or IL-1β, we were able to induce an inflammatory state, characterised by cytokine release (IL-8) and a decrease in trans-epithelial electrical resistance. Finally, we proved the applicability of the model in screening anti-inflammatory compounds by its reversibility. Using a well-known anti-inflammatory drug, TPCA-1, we were able to prevent cytokine-induced inflammation. This result was evident from the decreased secretion of IL-8 and retention of barrier function in treated cultures, similar to that observed in untreated cultures. Conclusion Overall, this complex 3D multicellular perfused intestine-on-a-chip model provides the versatile modularity of mimicking key features of intestinal inflammation and can, therefore, further support drug screening efforts and provide a platform for personalised medicine.

An adapted in vitro assay to assess Campylobacter jejuni interaction with intestinal epithelial cells: Taking into stimulation with TNFα

Campylobacter jejuni is the most prevalent foodborne bacterial infection agent. This pathogen seems also involved in inflammatory bowel diseases in which pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα), play a major role. C. jejuni pathogenicity has been extensively studied using in vitro cell culture methods, and more precisely “healthy” cells. In fact, no information is available regarding the behavior of C. jejuni in contact with TNFα-stimulated cells. Therefore, this research was designed to investigate the effect of TNFα on C. jejuni interaction with human intestinal epithelial cells (HT29 and HT29-MTX). To ensure IL-8 production induced by TNFα, human rtTNFα was added to HT29 and HT29-MTX before adhesion and invasion assays. About 108 CFU bacteria of C. jejuni strains cells were added to measure their adherence and invasion abilities using TNFα-stimulated cells versus non stimulated cells. Exposure to TNFα results in IL-8 overproduction by intestinal epithelial cells. In addition, the effect of TNFα pre-treatment on C. jejuni adhesion and internalization into eukaryotic cells is strain-dependent. Indeed, the adhesion/invasion process is affected in <50% of the strains tested when TNFα is added to the intestinal cells. Interestingly, TNFα affects more strains in their ability to adhere to and invade the mucus-secreting HT29-MTX cells. Among the 10 strains tested, the aero-tolerant C. jejuni Bf strain is one of the most virulent. These results suggest that the TNFα signalling pathway could participate in the internalization of C. jejuni in human intestinal cells and can help in understanding the pathogenicity of this microorganism in contact with TNFα-stimulated cells.

Saturated and Unsaturated Fatty Acids Differently Modulate Colonic Goblet Cells In Vitro and in Rat Pups1–3

Background: High-fat diets induce intestinal barrier alterations and promote intestinal diseases. Little is known about the effects of long-chain fatty acids (LCFAs) on mucin 2 (MUC2) production by goblet cells, which are crucial for intestinal protection.Objective: We investigated the effects of LCFAs on the differentiation of colonic goblet cells, MUC2 expression, and colonic barrier function.Methods: Upon reaching confluence, human colonic mucus-secreting HT29-MTX cells were stimulated (21 d) with a saturated LCFA (palmitic or stearic acid), a monounsaturated LCFA (oleic acid), or a polyunsaturated LCFA (linoleic, γ-linolenic, α-linolenic, or eicosapentaenoic acid). In addition, rat pups underwent oral administration of oil (palm, rapeseed, or sunflower oil) or water (10 μL/g body weight, postnatal days 10–15). Subsequently, colon goblet cells were studied by Western blotting, reverse transcriptase–quantitative polymerase chain reaction, and immunohistochemistry and colonic transmucosal electrical resistance was measured by using Ussing chambers.Results: In vitro, palmitic acid enhanced MUC2 production (140% of control) and hepatocyte nuclear factor 4α expression, whereas oleic, linoleic, γ-linolenic, α-linolenic, and eicosapentaenoic acids reduced MUC2 expression (at least −50% of control). All unsaturated LCFAs decreased the expression of human atonal homolog 1, a transcription factor controlling goblet cell differentiation (at least −31% vs. control). In vivo, rats fed palm oil had higher palmitic acid concentrations (3-fold) in their colonic contents and increased mucus granule surfaces in their goblet cells (>2-fold) than did all other groups. Palm oil also increased colonic transmucosal electrical resistance (245% of control), yet had no effect on occludin and zonula occludens-1 expression. In contrast, sunflower and rapeseed oils decreased goblet cell number when compared with control (at least −10%) and palm oil (at least −14%) groups.Conclusions: Palm oil in rat pups and palmitic acid in HT29-MTX cells increase the production of MUC2 and strengthen the intestinal barrier. In contrast, unsaturated LCFAs decrease MUC2 expression. These data should be taken into account in the context of preventive or therapeutic nutritional programs.

N-trimethyl chitosan chloride-coated PLGA nanoparticles overcoming multiple barriers to oral insulin absorption.

Although several strategies have been applied for oral insulin delivery to improve insulin bioavailability, little success has been achieved. To overcome multiple barriers to oral insulin absorption simultaneously, insulin-loaded N-trimethyl chitosan chloride (TMC)-coated polylactide-co-glycoside (PLGA) nanoparticles (Ins TMC-PLGA NPs) were formulated in our study. The Ins TMC-PLGA NPs were prepared using the double-emulsion solvent evaporation method and were characterized to determine their size (247.6 ± 7.2 nm), ζ-potential (45.2 ± 4.6 mV), insulin-loading capacity (7.8 ± 0.5%) and encapsulation efficiency (47.0 ± 2.9%). The stability and insulin release of the nanoparticles in enzyme-containing simulated gastrointestinal fluids suggested that the TMC-PLGA NPs could partially protect insulin from enzymatic degradation. Compared with unmodified PLGA NPs, the positively charged TMC-PLGA NPs could improve the mucus penetration of insulin in mucus-secreting HT29-MTX cells, the cellular uptake of insulin via clathrin- or adsorption-mediated endocytosis in Caco-2 cells and the permeation of insulin across a Caco-2 cell monolayer through tight junction opening. After oral administration in mice, the TMC-PLGA NPs moved more slowly through the gastrointestinal tract compared with unmodified PLGA NPs, indicating the mucoadhesive property of the nanoparticles after TMC coating. Additionally, in pharmacological studies in diabetic rats, orally administered Ins TMC-PLGA NPs produced a stronger hypoglycemic effect, with 2-fold higher relative pharmacological availability compared with unmodified NPs. In conclusion, oral insulin absorption is improved by TMC-PLGA NPs with the multiple absorption barriers overcome simultaneously. TMC-PLGA NPs may be a promising drug delivery system for oral administration of macromolecular therapeutics.

Vibrio vulnificus VvpE inhibits mucin 2 expression by hypermethylation via lipid raft-mediated ROS signaling in intestinal epithelial cells.

Mucin is an important physical barrier against enteric pathogens. VvpE is an elastase encoded by Gram-negative bacterium Vibrio vulnificus; however, the functional role of VvpE in intestinal mucin (Muc) production is yet to be elucidated. The recombinant protein (r) VvpE significantly reduced the level of Muc2 in human mucus-secreting HT29-MTX cells. The repression of Muc2 induced by rVvpE was highly susceptible to the knockdown of intelectin-1b (ITLN) and sequestration of cholesterol by methyl-β-cyclodextrin. We found that rVvpE induces the recruitment of NADPH oxidase 2 and neutrophil cytosolic factor 1 into the membrane lipid rafts coupled with ITLN to facilitate the production of reactive oxygen species (ROS). The bacterial signaling of rVvpE through ROS production is uniquely mediated by the phosphorylation of ERK, which was downregulated by the silencing of the PKCδ. Moreover, rVvpE induced region-specific methylation in the Muc2 promoter to promote the transcriptional repression of Muc2. In two mouse models of V. vulnificus infection, the mutation of the vvpE gene from V. vulnificus exhibited an increased survival rate and maintained the level of Muc2 expression in intestine. These results demonstrate that VvpE inhibits Muc2 expression by hypermethylation via lipid raft-mediated ROS signaling in the intestinal epithelial cells.

Biorelevant media resistant co-culture model mimicking permeability of human intestine

Cell culture models are currently used to predict absorption pattern of new compounds and formulations in the human gastro-intestinal tract (GIT). One major drawback is the lack of relevant apical incubation fluids allowing mimicking luminal conditions in the GIT. Here, we suggest a culture model compatible with biorelevant media, namely Fasted State Simulated Intestinal Fluid (FaSSIF) and Fed State Simulated Intestinal Fluid (FeSSIF). Co-culture was set up from Caco-2 and mucus-secreting HT29-MTX cells using an original seeding procedure. Viability and cytotoxicity assays were performed following incubation of FeSSIF and FaSSIF with co-culture. Influence of biorelevant fluids on paracellular permeability or transporter proteins were also evaluated. Results were compared with Caco-2 and HT29-MTX monocultures. While Caco-2 viability was strongly affected with FeSSIF, no toxic effect was detected for the co-cultures in terms of viability and lactate dehydrogenase release. The addition of FeSSIF to the basolateral compartment of the co-culture induced cytotoxic effects which suggested the apical mucus barrier being cell protective. In contrast to FeSSIF, FaSSIF induced a slight increase of the paracellular transport and both tested media inhibited partially the P-gp-mediated efflux in the co-culture. Additionally, the absorptive transport of propranolol hydrochloride, a lipophilic β-blocker, was strongly affected by biorelevant fluids. This study demonstrated the compatibility of the Caco-2/HT29-MTX model with some of the current biorelevant media. Combining biorelevant intestinal fluids with features such as mucus secretion, adjustable paracellular and P-gp mediated transports, is a step forward to more realistic in-vitro models of the human intestine.

Translocation of differently sized and charged polystyrene nanoparticles in in vitro intestinal cell models of increasing complexity

Intestinal translocation is a key factor for determining bioavailability of nanoparticles (NPs) after oral uptake. Therefore, we evaluated three in vitro intestinal cell models of increasing complexity which might affect the translocation of NPs: a mono-culture (Caco-2 cells), a co-culture with mucus secreting HT29-MTX cells and a tri-culture with M-cells. Cell models were exposed to well characterized differently sized (50 and 100 nm) and charged (neutral, positively and negatively) polystyrene NPs. In addition, two types of negatively charged NPs with different surface chemistries were used. Size strongly affected the translocation of NPs, ranging up to 7.8% for the 50 nm NPs and 0.8% for the 100 nm NPs. Surface charge of NPs affected the translocation, however, surface chemistry seems more important, as the two types of negatively charged 50 nm NPs had an over 30-fold difference in translocation. Compared with the Caco-2 mono-culture, presence of mucus significantly reduced the translocation of neutral 50 nm NPs, but significantly increased the translocation of one type of negatively charged NPs. Incorporation of M-cells shifted the translocation rates for both NPs closer to those in the mono-culture model. The relative pattern of NP translocation in all three models was similar, but the absolute amounts of translocated NPs differed per model. We conclude that for comparing the relative translocation of different NPs, using one intestinal model is sufficient. To choose the most representative model for risk assessment, in vivo experiments are now needed to determine the in vivo translocation rates of the used NPs.

Compound(s) secreted by Lactobacillus casei strain Shirota YIT9029 irreversibly and reversibly impair the swimming motility of Helicobacter pylori and Salmonella enterica serovar Typhimurium, respectively

We conducted experiments in order to examine whether the probiotic Lactobacillus casei strain Shirota YIT9029 (LcS) in vitro and in vivo antagonism of Helicobacter pylori and Salmonella, involves inhibition of the swimming motility of these pathogens. We report the irreversible inhibition of the swimming motility of H. pylori strain 1101 and reversible inhibition of Salmonella enterica serovar Typhimurium (S. Typhimurium) strain SL1344 by compound(s) secreted by LcS. In H. pylori 1101, irreversible inhibition results in the helical cells being progressively replaced by cells with 'c'-shaped and coccoid morphologies, accompanied by a loss of FlaA and FlaB flagellin expression. In S. Typhimurium SL1344, transient inhibition develops after membrane depolarization and without modification of expression of FliC flagellin. The inhibitory activity of strain LcS against both S. Typhimurium and H. pylori swimming motilities is linked with a small sized, heat-sensitive, and partially trypsin-sensitive, secreted compound(s), and needed the cooperation of the secreted membrane permeabilizing lactic acid metabolite. The inhibition of S. Typhimurium SL1344 swimming motility leads to delayed cell entry into human enterocyte-like Caco-2/TC7 cells and a strong decrease of cell entry into human mucus-secreting HT29-MTX cells.

Utilization of solid lipid nanoparticles for enhanced delivery of curcumin in cocultures of HT29-MTX and Caco-2 cells

Solid lipid nanoparticles (SLN) have shown potential for encapsulation, protection and delivery of lipophilic functional components. In this study, we have investigated the capabilities of SLN to deliver a hydrophobic polyphenol compound, curcumin, in a coculture system of absorptive Caco-2 and mucus secreting HT29-MTX cells. The cells were grown on transport filters to mimic the human intestinal epithelium. Because of the hydrophobic nature of curcumin, its delivery to the basolateral compartment is expected to take place via a paracellular route. The changes in curcumin concentration in various compartments (i.e., apical, basolateral, mucus, and cell lysates) were evaluated using fluorescence spectroscopy. Two SLN systems were prepared with different emulsifying agents. The encapsulation of curcumin in SLN caused enhanced delivery compared to unencapsulated curcumin. In addition, SLN showed enhanced delivery compared to emulsion droplets containing liquid soy oil. The SLN were retained on the apical mucosal layer to a greater extent than emulsion droplets. The presence of SLN did not affect the integrity of the cellular junctions, as indicated by the TEER values, and the route of transport of the solid particles was simple diffusion, with permeability rates of about 7 × 10(-6) cm s(-1). Approximately 1% of total curcumin was delivered to the basolateral compartment, suggesting that most of the curcumin was absorbed and metabolized by the cell.

Role of fibronectin-binding protein A in Clostridium difficile intestinal colonization

Clostridium difficile is a frequent cause of severe, recurrent, post-antibiotic diarrhoea and pseudomembranous colitis. Its pathogenicity is mediated mainly by two toxins, TcdA and TcdB. However, different adhesins have also been described as important colonization factors which are implicated in the first step of the intestinal infection. In this study, we focused our interest on one of these adhesins, fibronectin-binding protein A (FbpA), and on its role in the intestinal colonization process. A mutant of FbpA (CDΔFbpA) was constructed in C. difficile strain 630Δerm by using ClosTron technology. This mutant was characterized in vitro and in vivo and compared to the isogenic wild-type strain. Adhesion of the CDΔFbpA mutant to the human colonic epithelial cell line Caco-2 and to mucus-secreting HT29-MTX cells was examined. Surprisingly, the CDΔFbpA mutant adhered more than the wild-type parental strain. The CDΔFbpA mutant was also analysed in three different mouse models by following the intestinal implantation kinetics (faecal shedding) and caecal colonization (7 days post-challenge). We showed that in monoxenic mice, CDΔFbpA shed C. difficile in faeces at the same rate as that of the isogenic wild-type strain but its colonization of the caecal wall was significantly reduced. In dixenic mice, the shedding rate was slower for the CDΔFbpA mutant than for the isogenic wild-type strain during the first days of infection, but no significant difference was observed in caecal colonization. Similar rates of intestinal implantation and caecal colonization were observed for both strains in assays performed in human microbiota-associated mice. Taken together, our data suggest that FbpA plays a role in intestinal colonization by C. difficile.

Functional properties of lactic acid bacteria isolated from ethnic fermented vegetables of the Himalayas

A total of 94 strains of Lactic acid bacteria (LAB), previously isolated from ethnic fermented vegetables and tender bamboo shoots of the Himalayas, were screened for functional properties such as acidification capacity, enzymatic activities, degradation of antinutritive factors and oligosaccharides, production of biogenic amines, hydrophobicity and adherence to mucus secreting HT29 MTX cells. Strong acidification and coagulation activities of LAB strains were recorded. Most of the LAB strains showed antimicrobial activities against the used indicator strains; however, only Lb. plantarum IB2 (BFE 948) isolated from inziangsang, a fermented leafy vegetable product, produced a bacteriocin against Staphylococcus aureus S1. LAB strains showed enzymatic activities and also degraded oligosaccharides. Almost all the strains of LAB were non-producers of biogenic amines except few strains. Some strains of Lb. plantarum showed more than 70% hydrophobicity. Adherence to the mucus secreting HT29 MTX cells was also shown by seven strains indicating their probiotic nature.

Luminal leptin activates mucin-secreting goblet cells in the large bowel

Leptin has been suggested to be involved in tissue injury and/or mucosal defence mechanisms. Here, we studied the effects of leptin on colonic mucus secretion and rat mucin 2 (rMuc2) expression. Wistar rats and ob/ob mice were used. Secretion of mucus was followed in vivo in the rat perfused colon model. Mucus secretion was quantified by ELISA, and rMuc2 mRNA levels were quantified by real-time RT PCR. The effects of leptin alone or in association with protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) inhibitors on mucin secreted by human mucus-secreting HT29-MTX cells were determined. Leptin was detected in the rat colonic lumen at substantial levels. Luminal perfusion of leptin stimulates mucus-secreting goblet cells in a dose-dependent manner in vivo in the rat. Leptin (10 nmol/l) increased mucus secretion by a factor of 3.5 and doubled rMuc2 mRNA levels in the colonic mucosa. There was no damage to mucosa 24 h after leptin, but the number of stained mucus cells significantly increased. Leptin-deficient ob/ob mice have abnormally dense mucus-filled goblet cells. In human colonic goblet-like HT29-MTX cells expressing leptin receptors, leptin increased mucin secretion by activating PKC- and PI3K-dependent pathways. This is the first demonstration that leptin, acting from the luminal side, controls the function of mucus-secreting goblet cells. Because the gel layer formed by mucus at the surface of the intestinal epithelium has a barrier function, our data may be relevant physiologically in defence mechanisms of the gastrointestinal tract.

In vitro adherence and other properties of lactobacilli used in probiotic yoghurt-like products

This study compared selected probiotic properties of 9 strains of the Lactobacillus acidophilus group (6 L. acidophilus and 3 L. johnsonii) and 9 strains of the L. casei group, isolated from probiotic dairy products sold on the German market or supplied by a producer of starter cultures. Using an in vitro system simulating gastric transit, strains of the L. acidophilus group were more tolerant to the low pH 2.0 than strains of L. paracasei and L. rhamnosus which rapidly lost their viability after exposure to the simulated gastric juice containing pepsin. Milk showed a protective effect on strains less resistant to the gastric acidity. Bile salt hydrolase activity was detected in all strains of L. acidophilus/L. johnsonii but not in strains of the L. casei group. All lactobacilli were able to adhere to mucus secreting HT29 MTX cells and to human collagen type IV, fibrinogen and fibronectin although differences in the percentage of adhering bacteria were observed between different strains.

HT29-MTX/Caco-2 Cocultures as an in Vitro Model for the Intestinal Epithelium: In Vitro–in Vivo Correlation with Permeability Data from Rats and Humans

The diverse secretory and absorptive functions of the intestinal epithelium are conducted by a mixed population of absorptive cells and mucus-producing goblet cells as the major cell types. In order to approach the main characteristics in an in vitro model, a coculture system of absorptive Caco-2 cells and mucus-secreting HT29-MTX cells was developed and the permeability of a range of different drugs was tested. Variable goblet cell frequency can be achieved, preserving a significant barrier to drug transport and maintaining the differentiated features of both cell types. Absorption rates for actively transported drugs are rather underestimated in the cell culture model when compared to in vivo data. However, a good correlation with fraction absorbed in humans was attained separating the range of passively transported drugs into two groups of well-absorbable compounds with Peff ≥ 10 × 10−6 cm/s and drugs that are absorbed 40–70% with Peff=0.1–1 ×10−6 cm/s. A permeability of Peff<0.1 × 10−6 cm/s is suggested for low absorbable drugs.

Identification and characterization of adhesive factors of Clostridium difficile involved in adhesion to human colonic enterocyte‐like Caco‐2 and mucus‐secreting HT29 cells in culture

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 conducted. 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 adhesion of C. difficile strains was demonstrated by using rat polycolonal antibodies pAb 12 and pAb 27 directed against the 12 and 27 kDa proteins. Indeed, adhesion to Caco-2 cell monolayers of C. difficile strains grown in the presence of blood, without or with heat-shock, was blocked. Taken together, our results suggest that C. difficile may utilize blood components as adhesins to adhere to human intestinal cultured cells.

Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture.

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.