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Lactobacillus Paracasei CBA L74 prevents entrance of undigested gliadin peptides and rotavirus in Caco-2 cells Naples September 23-25 2014 Merlin Nanayakkara, Marco Sarno, Riccardo Troncone, Salvatore Auricchio and M.Vittoria Barone Vittori Buccirossi Roberto Nigro Department of Chemistry University of Napoli, Federico II,Italy Andrea Budelli Heinz. European Laboratory for the Investigation of Food Induced Diseases Department of Traslational Medical Science University of Napoli, Federico II,Italy Celiac disease Is a multifactorial disease caused by gluten ingestion in genetically susceptible subjects. The damage in the celiac intestine is mediated by an immune response both adaptive and innate, causing crypts hypertrofia and villus atrophy Diagnosis: antibodies anti TTG and anti endomisium Biopsy Therapy: Life long total abstinence from gluten containing food Other enviromental factors: Drugs (INF-alpha) and viral infections Some gliadin peptides are resistant to digestive enzimes (Mamone G. et al J Chromatography B, 855(2):236-241, 2007 P31-43 P57-68 Gliadin peptides dual activity Innate immunty PEPTIDE Prototype P31-43 LGQQQPFPPQQPY “T-CELL IMMUNOGENIC” PEPTIDES Prototype P57-68 QLQPFPQPQLPY Some gliadin peptides that are deamidated by tissue transglutaminase bind to typical CD HLA, DQ2 and/or DQ8 molecules, and induce an adaptive Th1 pro-inflammatory response (ie P56-68). Other gliadin peptides are able to initiate a response involving innate immunity independently from HLA interactions (ie P31-43) . Gliadin peptides enters into the cells by endocytosis Interaction of ‘toxic’ and ‘immunogenic’ A‐gliadin peptides with a membrane‐mimetic environment J of Molecular Recognition Vilasi s et al 2009 Caputo I. et al. Biochim Biophys Acta. 2010 LP CBA L74 effect on gliadin peptides entrance is concentration dependent Supernatant of LP CBA L74 effect on gliadin peptides entrance Supernatant of LP CBA L74 intereferes with endocytosis of dextran Cereals fermented with LP CBA L74 intereferes withgliadin peptides endocytosis Effect of LP CBA L74 supernatant on rotavirus (RV) entrance in RV-infected CaCo-2 cells No Infection Sup RV LP CBA L74 Sup + Fluorescence intensity CTRL RV Infection LP CBA L74 + RV RVInfection 18 16 14 12 10 8 6 4 2 0 N4 RV Infected LP CBA L74 +RV Infection Figure 1 Effect of LP CBA L74 supernatant on reactive oxygen species (ROS) in RV-infected Caco-2 cells 90 80 AIF/tot proteins 70 60 50 40 30 20 10 0 CTRL * p<.001 vs CTRL # p<.001 vs RV RV LP sup RV+LP sup Figure 2 Prof. Salvatore Auricchio ELFID Lab. Gliadin peptide P31-43 is similar to HRS (Hepatocyte growth factor-regulated tyrosine kinase substrate) Hrs is a key protein for the regulation of endocytic maturation Barone et al PloS One 2010, 2011 HRS has many binding partners. P31-43 is similar to a region of HRS needed for its correct localization to the endocytic vesicles p31-43 Clatrin binding domain P31-43 competes with HRS localisation Barone et al PloS One 2010 Delays maturation of early vesicles Delays endocytic vesicles dinamics Vesicles speed (µm in 10 min) .9 .8 .7 .6 .5 .4 .3 .2 .1 0 Caco2 cells EEA1/P31-43 30min EEA1/P31-43 3h LAMP 2/P31-43 3h Biposies P31-43-liss 30 min. 3h P56-68-liss 30 min. 3h Control CD EEA1/P31-43 3h P31-43 . Time lapse. CaCo2 cells treated with p3143 and P57-68 lissaminated. Vesicles containing P31-43 liss are slower that p5768 containing vesicles EEA1/P31-43 24 h Prolongs EGFR activation PTG Min at 37 C: 0 20’ 40’ 90’ 90’ P31-43 90’ 90’ EGFR WB: -EGFR EGFR -Tyr(P) ip: -EGFR Ab Barone et al GUT 2007 Gastroenterology 2007 Plos One 2010 Gliadin peptides can delay endocytic maturation and increse recycling vesicles IL-15 trans-presented Proliferation of epithelial cells Innate immunity M.V. Barone et al