Modeling the response of a biofilm to silver-based antimicrobial.

Biofilms are found within the lungs of patients with chronic pulmonary infections, in particular patients with cystic fibrosis, and are the major cause of morbidity and mortality for these patients. The work presented here is part of a large interdisciplinary effort to develop an effective drug delivery system and treatment strategy to kill biofilms growing in the lung. The treatment strategy exploits silver-based antimicrobials, in particular, silver carbene complexes (SCC). This manuscript presents a mathematical model describing the growth of a biofilm and predicts the response of a biofilm to several basic treatment strategies. The continuum model is composed of a set of reaction-diffusion equations for the transport of soluble components (nutrient and antimicrobial), coupled to a set of reaction-advection equations for the particulate components (living, inert, and persister bacteria, extracellular polymeric substance, and void). We explore the efficacy of delivering SCC both in an aqueous solution and in biodegradable polymer nanoparticles. Minimum bactericidal concentration (MBC) levels of antimicrobial in both free and nanoparticle-encapsulated forms are estimated. Antimicrobial treatment demonstrates a biphasic killing phenomenon, where the active bacterial population is killed quickly followed by a slower killing rate, which indicates the presence of a persister population. Finally, our results suggest that a biofilm with a ready supply of nutrient throughout its depth has fewer persister bacteria and hence may be easier to treat than one with less nutrient.

Antibody binding to a conformation-dependent epitope induces L-selectin association with the detergent-resistant cytoskeleton.

L-Selectin mediates leukocyte rolling on endothelium and immobilized leukocytes. Its regulation has been the subject of much study, and the conformation of the molecule may play an important role in its function. Here we report that a conformational change in L-selectin, induced by an anti-lectin domain mAb (LAM1-116) and recognized by another mAb directed to a conserved epitope on L-selectin (EL-246), predisposed L-selectin to cytoskeletal association. This effect was due to direct binding of the mAb, not to overt signaling events, and was specific to LAM1-116. Nineteen other anti-L-selectin mAbs directed against the lectin, epidermal growth factor, or short consensus repeat domains lacked this activity. The induced conformational change occurred at 37 degrees C, at 4 degrees C, in the presence of sodium azide and tyrosine kinase inhibitors herbimycin A and genistein, and with soluble detergent-extracted L-selectin. In the presence of LAM1-116, EL-246 induced cytoskeletal association of L-selectin in the absence of Ab cross-linking as visualized by L-selectin staining after low dose detergent treatment of the cells. We propose that the conformational change described herein regulates L-selectin-mediated events by exposing a high avidity binding site that, when engaged, triggers association of L-selectin with the cytoskeleton, which may lead to stronger tethers with physiological ligands.

Comparative ultrastructure and expression of L-selectin on bovine alphabeta and gammadelta T cells.

Compared to most alphabeta T cells, bovine gammadelta T cells express two to five times the level of L-selectin and have a much higher capacity to roll on monolayers of endothelial cells, platelets, and leukocytes in assays done under physiological flow. To gain additional insight into the basis for these differences, scanning (SEM) and transmission (TEM) electron microscopy were used to compare the cellular ultrastructure of bovine gammadelta and alphabeta T cells and to study the expression of L-selectin on these cells. It is interesting that gammadelta T cells had more than twice as many microvilli and other surface projections on a per cell basis as alphabeta T cells. This was not due to the gammadelta T cell being larger; after adhesion and fixation procedures used for the EM studies, gammadelta T cells averaged 3.9 +/- 0.01 microm in diameter, whereas alphabeta T cells were slightly larger (5.7 +/- 0.01 microm in diameter). As previously shown for human neutrophils and lymphocytes, L-selectin was preferentially localized to the tips of the microvilli. In contrast, WC1, a lineage-specific antigen on gammadelta T cells, was localized on the plasmalemma between the microvilli. Our findings suggest that more effective rolling of gammadelta T cells in various in vitro flow assays may be due to the greater number of microvilli on gammadelta T cells leading to a higher number of contact sites during adhesion events. In addition, this physical parameter may explain the increased level of L-selectin expression on gammadelta versus alphabeta T cells because L-selectin is clustered at the tips of microvilli.