Pati M. Glee

Select Plant Tannins Induce IL-2Rα Up-Regulation and Augment Cell Division in γδ T Cells

γδ T cells are innate immune cells that participate in host responses against many pathogens and cancers. Recently, phosphoantigen-based drugs, capable of expanding γδ T cells in vivo, entered clinical trials with the goal of enhancing innate immune system functions. Potential shortcomings of these drugs include the induction of nonresponsiveness upon repeated use and the expansion of only the Vδ2 subset of human γδ T cells. Vδ1 T cells, the major tissue subset, are unaffected by phosphoantigen agonists. Using FACS-based assays, we screened primary bovine cells for novel γδ T cell agonists with activities not encompassed by the current treatments in an effort to realize the full therapeutic potential of γδ T cells. We identified γδ T cell agonists derived from the condensed tannin fractions of Uncaria tomentosa (Cat’s Claw) and Malus domestica (apple). Based on superior potency, the apple extract was selected for detailed analyses on human cells. The apple extract was a potent agonist for both human Vδ1 and Vδ2 T cells and NK cells. Additionally, the extract greatly enhanced phosphoantigen-induced γδ T cell expansion. Our analyses suggest that a tannin-based drug may complement the phosphoantigen-based drugs, thereby enhancing the therapeutic potential of γδ T cells.

Influence of cell surface hydrophobicity on attachment of Candida albicans to extracellular matrix proteins

Cell surface hydrophobicity expression by Candida albicans facilitates a diffuse binding distribution of yeast cells to host tissues ex vivo. One possibility for the receptor site responsible for the binding pattern of hydrophobic cells is the extracellular matrix (ECM). In this study, we evaluated the interaction of hydrophobic and hydrophilic C. albicans with ECM proteins immobilized onto wells of microtitre tissue. Culture plates, and the ability of ECM proteins to block the binding of hydrophobic cells to splenic tissue ex vivo. Hydrophobic C. albicans bound in greater numbers than hydrophilic cells to the immobilized proteins, particularly fibrinogen, fibronectin, collagen type IV and laminin. Similar results were obtained regardless of C. albicans strains or of growth medium. Collagen and fibronectin blocked the binding of hydrophobic cells to the white pulp but not to the marginal zones in splenic tissues when tested with the ex vivo assay. These results suggests that the diffuse binding pattern of hydrophobic cells in the ex vivo assay may be due to their enhanced ability over hydrophilic cells to bind to ECM proteins, particularly fibronectin and collagen type IV.

Inhibition of hydrophobic protein-mediated Candida albicans attachment to endothelial cells during physiologic shear flow.

ABSTRACT Adhesion interactions during hematogenous dissemination ofCandida albicans likely involve a complex array of host and fungal factors. Possible C. albicans factors include changes in cell surface hydrophobicity and exposed antigens that have been shown in static adhesion assays to influence attachment events. We used a novel in vitro shear analysis system to investigate host-pathogen interactions and the role of fungal cell surface hydrophobicity in adhesion events with human endothelial cells under simulated physiologic shear. Endothelial monolayers were grown in capillary tubes and tested with and without interleukin-1β activation in buffered medium containing human serum. Hydrophobic and hydrophilic stationary-phase C. albicans yeast cells were infused into the system under shear flow and found to adhere with widely varying efficiencies. The average number of adherent foci was determined from multiple fields, sampled via video microscopy, between 8 and 12 min after infusion. Hydrophobic C. albicans cells demonstrated significantly more heterotypic binding events (Candida-endothelial cell) and greater homotypic binding events (Candida-Candida) than hydrophilic yeast cells. Cytokine activation of the endothelium significantly increased binding by hydrophobic C. albicans compared to unactivated host cells. Preincubation of hydrophobic yeast cells with a monoclonal antibody against hydrophobic cell wall proteins significantly blocked adhesion interactions with the endothelial monolayers. Because the antibody also blocks C. albicans binding to laminin and fibronectin, results suggest that vascular adhesion events with endothelial cells and exposed extracellular matrix may be blocked during C. albicans dissemination. Future studies will address the protective efficacy of blocking or redirecting blood-borne fungal cells to favor host defense mechanisms.

Presence of multiple laminin- and fibronectin-binding proteins in cell wall extract of Candida albicans: influence of dialysis

Candida albicans has been reported to express only one to three proteins that bind extracellular matrix proteins, such as laminin and fibrinogen. In those reports, cell wall extracts were subjected to various processing steps, such as dialysis and lyophilization, prior to Western blot analysis. Here, we demonstrate that dialysis for only 2 h of cell wall protein extracts results in a substantial loss (40-60%) of protein. With overnight dialysis, the loss was increased further. After 2 h of dialysis, wall extracts contained fewer laminin- and fibronectin-reactive proteins. In addition, the number of wall proteins in the extracts detected by a polyclonal anti-human fibronectin receptor antiserum decreased after dialysis. These results demonstrate that the C. albicans yeast cell wall contains multiple proteins capable of binding laminin and fibronectin and many of these proteins are not functionally detectable following dialysis.

Aggregation of hydrophobic cell wall proteins of Candida albicans

Abstract The exterior surface of the cell wall of Candida albicans can be hydrophobic or hydrophilic. Hydrophobic cell wall proteins of masses 32–40 kDa from C. albicans demonstrated strong aggregation tendencies which influenced both preparation and analysis. The proteins aggregated at 37°C in water and various buffers. Aggregation of the isolated proteins increased with time, was rapidly promoted by heat (3 min, 100°C), and was influenced by dialysis method, protein concentration, and centrifugal ultrafiltration. The presence of 2% sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol (2-ME) inhibited aggregate formation, but heating in SDS and 2-ME did not disrupt pre-formed aggregates. Aggregate sizes in acrylamide gels corresponded to roughly dimer and trimer molecular weights. Electron micrographs of negatively-stained hydrophobic protein samples demonstrated very large aggregates which were not observed in unfractionated cell wall protein samples. The potential impact of hydrophobic protein aggregation upon the ectomural fibrillar structure of the C. albicans cell wall is discussed.

[34] Adhesion of fungi

Publisher Summary Adhesion of a fungus to an appropriate surface precipitates a variety of biological events that could influence fungal survival and could ultimately jeopardize the viability or integrity of the host surface. Following adhesion, fungal spores elaborate vegetative growth structures or produce structures that penetrate the host surface. Understanding the mechanisms of fungal adhesion to inanimate and animate structures is tantamount to the development of effective modalities for inhibiting (or enhancing) successful fungal colonization. This chapter describes the development of fungal adhesion assays along with details of two convenient assays. The general designs of the adhesion assays closely mimic those used for studying bacterial adhesion. Bacterial adhesion assays may translate to fungal systems relatively well. Caveats that must be considered when applying bacterial adhesion assay designs to fungi are also discussed in the chapter. One common practice in preparing cells for adhesion assays is to fix the cells with glutaraldehyde, formalin, or other similar agent, or to kill the cells with a chemical agent. Although this practice provides a means to prepare a common, convenient source of cells and prevents cell growth during the adhesion assay, it results in alterations in the cell surface that may influence the outcome of the experiment.