Sept 27, 2012.rtf

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University of Toronto St. George
Molecular Genetics and Microbiology
Richard Brown

MGY440 - September 27, 2012 Adenovirus receptor interaction can be anywhere on cell - top surface, bottom surface, etc. - done in studies with cell culture - usually without polarized cells Polarized cells = distinct sides (have apical surface, basal-lateral side) Intestinal epithelium, airway epithelium, etc. - healthy epithelium = intact Confluent cells in culture - may still have tiny spaces even though appear confluent whereas in reality, intact epithelial layer, with zero spaces (tight junctions, etc.) When cell is polarized, no integrins, no CAR on surfce - integrins on bottom, CAR part of tight junction (between cells, adhesion molecule helping hold cells together) - in human body's airway epithelium, no exposed receptor - good for us - but how are abrasions there? How does the infection establish itself? - abrasions can occur on for instance hands, because they are constantly used - however how are abrasions found in the lungs? LUTSCHG PAPER - macrophages (alveolar macrophages) take up viruses etc. - host defense - FIGURE 1 - polarized cells - want to investigate all different surfaces - so need cells to be NOT Stuck to plastic - use transwell - cells growing on insert, which has pores (of different sizes) - diffusion freely back and forth through the pores of insert - once cells able to form tight junctions (polarized layer) what is added above the cells, the only way to get to below insert is via transportion through the cell (vice versa) - - two differen cell types - open bars are A549 cells, black bars are bronchial epithelial cells (different part of airway) - non polarized = soon after seeding (cells not yet polarized because there are spaces between there); polarized = confluent and having established tight junctions - AP = infecting from apical surface; BL = basal lateral surface - EDTA = chelates calcium, which is necessary for adhesion - cannot assess infection by CPE because cannot see the cells once seeded in Transwell containers - instead, used reporter adenovirus type 5, that carries gene for murine interleukin 2 - if virus is successful in delivering genome to nucleus, IL2 secreted from cell and can be assayed in culture fluid - - infects non-polarized cells better - polarized cells - adding virus from apical surface - hardly any infection - polarized cell - infect form basal lateral surface - more cells infected, thus more IL2 in culture fluid - polarized cell - add EDTA and infecting from apical surface - opened up tight junctions - like as if added from basal lateral surface - B section - flow cytometry - measure fluorescence - cells in suspension, so this tells you what is on the surface, but doesn't specify WHICH suface - percentage of cells with signal to the receptors on surface - resistance to infection in APICAL without EDTA - these molecules are present, so not due to no molecules - C section - co-culture - add macrophages to the top of transwells - magnified section = has virus - count of virions per cell to generate D - D section - more virions in macrophages than epitheliel cells - E section - looking at successful genome delivery - looking at presence of particle in cell - productive infection of epithelial cell in presence and absence of - add macrophages - similar to if infecting from basal lateral surface Supplementary figure 2 - is virus replicating in macrophages? - IL2 made by macrophages or epitheial cells or both? - macrophages are taking up virus but not delivering its genome to macrophage nucleus Figure 2 - still Lutschg paper - if macrophages secreting factor, which should be in medium - then use this medium to treat epithelial cells - then infect with adenovirus with reporter gene for IL2 - incubate, wait - measure production of IL2 - in B, looking at amount of IL2 produced - infecting from top, no culture medium, little IL2 produced - consistent - infect from apical surface with culture medium, much more IL2 produced - in both cell lines - in C - time course experiment - takes up to four hours to get enough of macrophage's secreted factor - in D - add macrophage medium to either apical and/or basal - most when adding from both apical and basal surfaces - in E - measure electrical resistence across cells - transepithelial resistence increases as cells become confluent, indicating tight junctions are intact - - add EDTA - destroys tight junctions - spaces between cells, and reistence falls - although conditioned medium makes epithelial cells susceptible to infection from apical surface NOT because opening tight junctions because NOT CHANGING THE RESISTENCE - in F - confocal microscopy is UV fluorescence - bronchial epithelial cells - no macrophages - on left, control - virus labeled with red fuorescent dye - beta cateinen marker for lateral surface; blue is nuclei - incubation with conditioned medium better binding of labelled virus - get better susceptibility to infection of apical surface - shows binding at apical surface - tight junctions remain intact - - in G - most of virus signal is at the apical surface - virus doesn't have access to basal-lateral surface - as result of cells exposed to conditioned medium, virus is binding to apical surface and tight junctions remain intact Figure 3 - what is the fact in conditioned medium? - CXCL8 - IL8 - in B - neutralize IL8 with antibody against it - reduces effect of IL8 - suggesting IL8 is in conditioned medium that is having an effect - antibody against IL10, no difference - in C - used antibody to block one receptor of IL8, and also block othe receptor - has an effect - antibody against receptors of IL8 - reduces the effect - thus suggesting IL-8 is important - IL8 probably not the only factor secreted by macrophages - in E, the two IL8 receptors are apical surface receptors - Figure 4? - integrins at basal surface - attaching cells to surface - treat with conditioned medium, alpha v beta three goes from basal to apical surface - same effect with treatment of IL8 by itself Zo1 = marker for
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