Comment[ArrayExpressAccession]	E-MEXP-3445
Investigation Title	Differential Targeting of the E-Cadherin and b-Catenin Complex by Gram-positive Probiotic Lactobacilli Improves Epithelial Barrier Function
Comment[Submitted Name]	Differential Targeting of the E-Cadherin and b-Catenin Complex by Gram-positive Probiotic Lactobacilli Improves Epithelial Barrier Function
Comment[AEExperimentDisplayName]	Differential Targeting of the E-Cadherin and b-Catenin Complex by Gram-positive Probiotic Lactobacilli Improves Epithelial Barrier Function
Comment[MIAMExpressLogin]	ZMBE-IfI
Comment[MIAMExpressSubmissionID]	7941
Experiment Description	The intestinal ecosystem is balanced by dynamic interactions between resident and incoming microbes, the gastrointestinal barrier, and the mucosal immune system. However, in the context of inflammatory bowel diseases (IBD) where the integrity of the gastrointestinal barrier is compromised, resident microbes contribute to the development and perpetuation of inflammation and disease. In this context, probiotic bacteria exert beneficial effects enhancing epithelial barrier integrity. However, the mechanisms underlying these beneficial effects are only poorly understood. Here, we comparatively investigated the effects of four probiotic lactobacilli, namely L. acidophilus, L. fermentum, L. gasseri, and L. rhamnosus in a T84 cell epithelial barrier model. Results of DNA-microarray experiments indicating that lactobacilli modulate the regulation of genes encoding in particular adherence junction proteins such as E-cadherin and b-catenin were confirmed by qRT-PCR. Furthermore, we show that epithelial barrier function is modulated by Gram-positive probiotic lactobacilli via their effect on adherence junction protein expression and complex formation. In addition, incubation with lactobacilli differentially influences the phosphorylation of adherence junction proteins and of PKC isoforms such as PKCd which thereby positively modulates epithelial barrier function. Further insight into the underlying molecular mechanisms triggered by these probiotics might also foster the development of novel strategies for the treatment of gastrointestinal diseases (e.g. IBD).
Experimental Design	in_vitro_design	co-expression_design	time_series_design	pathogenicity_design
Comment[AEExperimentType]	transcription profiling by array
Experimental Factor Name	Incubate	IncubationTime
Experimental Factor Type	growth_condition	time
Person Last Name	Cichon
Person First Name	Christoph
Person Email	cichon@uni-muenster.de
Person Phone	+49 251 8352130
Person Fax	+49 251 8356467
Person Affiliation	Center for Molecular Biology of Inflammation
Person Address	Institute for Infectiology, Von-Esmarch-Strasse 56, Muenster, NRW, 48149, Germany
Person Roles	submitter
Person Roles Term Source REF	MGED Ontology
Quality Control Type
Public Release Date	2012-02-10
Comment[ArrayExpressSubmissionDate]	2011-11-23 05:04 PM
Publication Title	Differential Targeting of the E-Cadherin/β-Catenin Complex by Gram-Positive Probiotic Lactobacilli Improves Epithelial Barrier Function
Publication Status	submitted
Protocol Name	P-MTAB-24190	P-MTAB-24191	P-MTAB-24192	P-MTAB-24193	P-MTAB-24194	P-MTAB-24195	P-MTAB-24196
Protocol Description	Total RNA was extracted using the RNeasy Minikit (Qiagen, Hilden, Germany) and cRNA was prepared as described previ- ously (Thykjaer et al., 2001). Briefly, RNA was isolated from tissue culture flasks. Total RNA samples were used to generate biotinylated cRNA targets according to the “Affymetrix Microarray Suite 4.0 User Guide”. Enzymes were supplied by Invitrogen (Breda, the Netherlands) and Roche (Mannheim, Germany). The oligo-dT primer containing a T7 RNA polymerase promoter was purchased from Ambion (Huntington, UK).<br>(Parameters: Extracted product = total_RNA, Amplification = none)	Hybridization and scanning to U133 Plus 2.0 chip sets (Affymetrix, Santa Clara, CA) according to the supplier’s instructions.<br>(Parameters: Scanning hardware = 418 [Affymetrix], Scanning software = MAS/GCOS/GREX [Affymetrix])	bacteria: Luria-Bertani broth T84 (ECACC, Salisbury, UK, passage 59-71) was grown in a 5% CO2humidified incubator at 37 degrees C with medium containing Ham’s F-12 nutrient mixture and DMEM (PAA Laboratories, C�lbe, Germany) supplemented with 10% FCS, antibiotics (PenStrep) (Madara et al., 1987)<br>(Parameters: start time = 0, stop time = 180, time unit = minutes, min temperature = 37, temperature unit = C, media = DMEM/F-12/LB)	Labelled cRNA was prepared by using the “Microarray Target Synthesis Kit” and biotin labelled UTP from Roche (Mannheim, Germany) and hybridized to Hu U95A chip sets (representing 12 000 human DNA sequences, Affymetrix, Santa Clara, CA) according to the supplier’s instructions.<br>(Parameters: Amount of nucleic acid labeled = 5, Amplification = none, Mass unit = Micro gram)	Affymetrix chp protocol	Overnight cultures of EcN and of EPEC strain E2348/69, grown in Luria�Bertani broth, were diluted (1:33) in serum- and antibiotic-free tissue culture medium containing 0.5% mannose and grown at 37degrees C to mid-log growth phase. Epithelial cell mono-layers were infected as previously described (Tomson et al., 2004) with a moi of 100. In co-infection experiments with EPEC and EcN, both strains were present in a ratio of 1:1 at a combined moi of 100.	Hybridization and staining was done according to the “Affymetrix Microarray Suite 4.0 User Guide”.<br>(Parameters: Chamber type = Affymetrix- GeneChip Hyb Oven 640, Quantity of label target used = 1, Mass unit = Micro gram, time = 60, Tiny time unit = minutes, Volume = 100, Volume unit = Micro litre, temperature = 45)
Protocol Type	nucleic_acid_extraction	image_acquisition	grow	labeling	bioassay_data_transformation	specified_biomaterial_action	hybridization
Protocol Term Source REF	MGED Ontology	MGED Ontology	MGED Ontology	MGED Ontology	MGED Ontology	MGED Ontology	MGED Ontology
Term Source Name	MGED Ontology
Term Source File	http://mged.sourceforge.net/ontologies/MGEDontology.php
SDRF File	E-MEXP-3445.sdrf.txt
PubMed ID	22179242
Publication Author List	Hummel, Stephanie; Veltman, Katharina; Cichon, Christoph; Sonnenborn,Ulrich; Schmidt, M. Alexander
Publication DOI	10.1128/AEM.06983-11