Role of the glycocalyx in regulating access of microparticles to apical plasma membranes of intestinal epithelial cells: implications for microbial attachment and oral vaccine targeting.

Abstract

SummaryTransepithelial transport of antigens and pathogens across the epithehal barrier by M cells may be a prerequisite for induction ofmucosal immunity in the intestine. Efficient transport of antigens and pathogens requires adherence to M cell apical surfaces. Couphng of antigen-containing particles to the pentameric binding subunit of cholera toxin (CTB) has been proposed as a means for increasing antigen uptake because the CTB receptor, ganglioside GM1, is a glycohpid present in apical membranes of all intestinal epithehal cells. To test the accessibility of enterocyte and M cell membrane glycohpids to ligands in the size ranges of viruses, bacteria, and particulate mucosal vaccines, we analyzed binding of CTB probes of different sizes to rabbit Peyer's patch epithelium. Soluble CTB-fluorescein isothiocyanate (diameter 6.4 nm) bound to apical membranes of all epithelial cells. CTB coupled to 14 nm colloidal gold (final diameter, 28.8 nm) failed to adhere to enterocytes but did adhere to M cells. CTB-coated, fluorescent microparticles (final diameter, 1.13 p~m) failed to adhere to enterocytes or M cells in vivo or to well-differentiated Caco-2 intestinal epithelial cells in vitro. However, these particles bound specifically to GM1 on BALB/c 3T3 fibroblasts in vitro and to undifferentiated Caco-2 cells that lacked brush borders and glycocalyx. Measurements of giycocalyx thickness by electron microscopy suggested that a relatively thin (20 nm) glycocalyx was sufficient to prevent access of 1-~m microparticles to glycolipid receptors. Thus, the barrier function of the intestinal epithelial cell glycocalyx may be important in limiting microbial adherence to membrane glycolipids, and in CTB-mediated targeting of vaccines to M cells and the mucosal immune system.

Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination

“…Furthermore, Western blot analysis under nonreducing conditions revealed that CTB expressed in rice formed a pentamer with 55 to 65 kDa (Fig. 1F), indicating that most of the CTB expressed in rice seed is considered to be a functionally native form possessing the ability to bind to the GM1 ganglioside, known to be expressed at the apical surface of the intestinal epithelium and to be a receptor for CTB (28).…”

Breast milk, microbiota, and intestinal immune homeostasis

“…He suggests that not only do intestinal bacteria stimulate lymphoid elements, but they also influence components of the single layer of epithelium that makes up the mucosal barrier by stimulating the development of microvilli (19,20) and tight junctions (21). These bacteria also activate the release of mucin from epithelial goblet cells to form the glycocalyx as a physical and antibacterial barrier (19)(20)(21)(22). In addition, he provides evidence that intestinal bacteria can stimulate epithelial cells and Paneth cells to release antibacterial peptides into the glycocalyx and lumen which helps to regulate the composition of colonizing intestinal bacteria (19,23).…”

Extracellular, cell-permeable survivin inhibits apoptosis while promoting proliferative and metastatic potential

“…However, survivin recombinant protein (BIRC5) at the same concentrations was not endocytosed (Figure 6). Transferrin (Sandvig and van Deurs, 1990) and CTB (Gizurarson et al, 1992;Frey et al, 1996; , MCF-7, Panc1, PC3, SaOS2, U2OS, HCC, and HBMS cells were cultured with control-or Surv-WT-conditioned medium for 24 h. Cells were fixed and stained with antibodies to HA, magnification  1000. (B) Western blot analysis using antibodies to survivin shows the retarded B18 kDa band of the Flag-HA-survivin fusion protein only in those lysates from the cells grown in conditioned medium.…”