Interaction of Staphylococcus epidermidis with endothelial cells in vitro.

Staphylococcus epidermidis is a leading cause of nosocomial bacteremia, yet virtually nothing is known about how this pathogen interacts with human endothelial cells. We present evidence here that two biofilm-producing strains of S. epidermidis adhere to two types of endothelial cell lines in vitro and that adherence is significantly increased after briefly heat-treating the bacteria at 40 degrees C in the presence of calcium. This mild heat treatment resulted in bacteria that were 5 to more than 20 times more adherent than untreated controls. While the adherence of bacteria in all phases of growth was increased after heat treatment, heat-treated late stationary phase cells were generally the most adherent. Electron microscopy demonstrated that S. epidermidis was internalized and appeared to exist free in the cytoplasm. Adherence to endothelium, should it occur in vivo during bacteremia, may be a virulence factor associated with this bacterium's pathogenesis.

Characterization of a monoclonal antibody that binds to an epitope on soluble bacterial peptidoglycan fragments.

We employed an inhibition-type enzyme-linked immunosorbent assay (ELISA) to characterize a murine immunoglobulin M monoclonal antibody (MAb) that bound soluble macromolecular peptidoglycan (PG). With this ELISA, the MAb was capable of detecting soluble PG concentrations of less than 10 ng/ml. Enzymatic digestion of PG reduced binding by more than 100-fold, implying that the epitope recognized by this antibody depended on repeating subunits within the glycan backbone. Additionally, the MAb bound to epitopes on both O-acetylated and non-O-acetylated PG fragments from gram-negative bacteria, as well as PG fragments from Staphylococcus aureus and PG fragments released into the medium by a number of gram-positive and gram-negative bacteria.

The effects of Cryptococcus neoformans-secreted antigens on tumor necrosis factor-alpha-induced intercellular adhesion molecule-1 expression on human lung epithelial cells.

Since primary infection with Cryptococcus neoformans usually occurs in the lungs, and since pulmonary cryptococcosis involves interactions between yeasts and alveolar epithelial cells, we have begun to study the effects of C. neoformans and its secreted antigens (SA) on epithelial reactions potentially associated with localized inflammation. We report here that SAs from encapsulated and acapsular strains of C. neoformans caused significant reductions in tumor necrosis factor-alpha (TNF-alpha)-induced intercellular adhesion molecule-1 (ICAM-1) expression on A549 lung epithelial cells in culture. We also present evidence that the reduction in ICAM-1 expression was not associated with SA-induced shedding of this adhesion molecule.

An opsonizing monoclonal antibody that recognizes a noncapsular epitope expressed on Cryptococcus neoformans.

A mouse hybridoma secreting a monoclonal antibody (MAb) that bound a noncapsular epitope expressed on C. neoformans was developed by immunizing BALB/c mice with formalin-killed serotype A yeasts. The hybridoma, designated CSFi, secreted an immunoglobulin G2b MAb that reacted with all C. neoformans serotypes tested, including the acapsular mutant ATCC 52817 (Cap67). Postsectioned immune electron microscopy revealed extensive binding of the MAb to the cell walls of both encapsulated and acapsular yeasts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of secreted antigens recovered from concentrated culture supernatants from both encapsulated and acapsular strains was conducted. The results showed that this MAb bound predominantly to antigens with molecular masses of approximately 75 and 100 kDa. A competitive enzyme-linked immunosorbent assay was used to demonstrate that the MAb was not cross-reactive with purified glucuronoxylomannan derived from either serotypes A or D. Experiments conducted with mouse peritoneal phagocytes and the mouse phagocyte-like cell line, J774A.1, demonstrated that the CSFi MAb opsonized the yeasts and increased their adherence to both types of phagocytic cells. We conclude, therefore, that antibodies directed at noncapsular epitopes can serve as opsonins and may have a role in modulating cryptococcal infection.

The in vitro interaction of Cryptococcus neoformans with human lung epithelial cells.

The interaction of Cryptococcus neoformans with a human lung epithelial cell line (A549) is described. Encapsulated and acapsular strains adhered to epithelial cells in a time-dependent manner, with the acapsular strain being the most adherent under all conditions tested. Internalized cryptococci were additionally observed. The expression of the adhesins responsible for adherence to the epithelial cells was induced by growth at 37 degrees C. Adhesin expression was repressed in all strains by growth with sucrose as the sole carbon source. A strain-specific repression of adhesin expression was observed after growth with galactose and xylose. A variety of carbohydrates included in the assay suspensions blocked adherence, implicating certain carbohydrate moieties that might serve as ligands for the yeast adhesin. Finally, a monoclonal antibody is described that inhibited cryptococcal adherence to the epithelial cells. Collectively, the results demonstrate a specific interaction between C. neoformans and lung epithelial cells mediated by yeast adhesins whose expression is regulated by environmental factors.

Reduced recovery of a Cryptococcus neoformans adherence mutant from a rat model of cryptococcosis.

Stable mutants of Cryptococcus neoformans (strain CSF-1) induced by treatment with ultraviolet light and nitrosoguanidine were isolated that demonstrated reduced adherence to glial cells in culture. Adherence of the mutants, as measured by a radiometric assay, was reduced by 50-70% of that attained for the parent CSF-1 strain. The adherence mutants appeared to be phenotypically similar to the CSF-1 strain. However, all but one mutant (designated as CSF-23) demonstrated slightly slower growth rates than the wild-type strain. The CSF-1 and CSF-23 strains were injected intravenously and intratracheally into normal rats and rats immunosuppressed by cyclophosphamide treatment, and the organ distribution and recovery of viable yeasts determined over 2-96 h. During this relatively short period of observation the majority of the yeasts were localized in the lungs. By either route of injection, the recovery of the CSF-23 adherence mutant was reduced by as much as 90% of that obtained for the wild-type strain. The results indicated that host cell adherence may be important for the persistence of cryptococci in tissue and that further studies with the adherence mutants are warranted.

Comparisons between in vitro glial cell adherence and internalization of non-encapsulated and encapsulated strains of Cryptococcus neoformans.

The adherence of non-encapsulated and encapsulated strains of Cryptococcus neoformans to rat glial cells in culture was compared. Like the encapsulated strain, the adherence of the unencapsulated strain was affected by the yeast culture age and growth temperature. Yeasts grown to late stationary phase at 37 degrees C were the most adherent. Neither encapsulated nor non-encapsulated strains adhered to glial monolayers when the experiments were conducted at 5 degrees C, indicating that metabolically active mammalian cells were required for yeast adherence. Additionally, the non-encapsulated strain was consistently three times more adherent than the encapsulated strain, suggesting that the non-encapsulated strain either had more adhesins or more adhesins were exposed. Electron microscope studies indicated that both strains were internalized by glial cells, an event previously not reported for this mammalian cell type. The same carbohydrates (N-acetyl-D-glucosamine, sucrose and inositol) that inhibited adherence of the encapsulated strain the most, also inhibited adherence of the non-encapsulated strain, again indicating similar adhesin mechanisms. Both encapsulated and non-encapsulated strains were made non-adherent by treatment with pronase, papain, trypsin and amylase. Immobilized amylase appeared to remove an adhesin fragment that bound to glial cells and inhibited the adherence of intact cryptococci.

Conditions affecting the adherence of Cryptococcus neoformans to rat glial and lung cells in vitro.

Conditions affecting the adherence of clinical isolates of Cryptococcus neoformans to rat glial and lung cell cultures were studied. Adherence to glial cells was a time-dependent process that was affected by the yeast culture age and growth temperature. The most adherent yeasts were those from 48 h cultures grown at 37 degrees C. Formalin-treating the yeasts did not affect adherence but formalin-treating the glial monolayers prevented yeast binding. Treating the yeasts with trypsin reduced adherence to glial monolayers, indicating that the yeast adhesin had a trypsin-labile protein component. Certain carbohydrates inhibited cryptococcal adherence to glial and lung cells in a time and concentration-dependent manner. Of the carbohydrates tested, N-acetyl-D-glucosamine, sucrose, lactose, sorbitol and myo-inositol were the most inhibitory, while mannose, galactose and xylose were the least inhibitory. The results collectively indicated that the mechanisms of adherence of C. neoformans to lung cells were similar to those of glial cells and that both involved a protein-containing adhesin on the cryptococcal surface that was expressed only after growth at 37 degrees C. Carbohydrate receptors also appeared to be involved with these interactions.

Enhanced expansion of the thymic CD8+ cell subset as a potential mechanism for the generation of enhanced antitumor cytotoxicity by thymocytes from low-dose melphalan-treated MOPC-315 tumor bearers.

We have previously shown that thymocytes from low-dose melphalan (L-phenylalanine mustard)-treated MOPC-315-tumor-bearing mice (melphalan TuB) are able to generate an enhanced level of anti-MOPC-315 cytotoxicity, as compared to thymocytes from untreated MOPC-315-tumor-bearing mice or thymocytes from untreated or low-dose melphalan-treated normal mice, upon in vitro stimulation with MOPC-315 tumor cells in the presence of a low concentration of recombinant interleukin-2 (rIL-2). Here we show that the generation of enhanced anti-MOPC-315 cytotoxicity by melphalan TuB thymocytes depends on the ability of the thymocytes to proliferate. In addition, the ability of melphalan TuB thymocytes to generate an enhanced level of anti-MOPC-315 cytotoxicity correlated with their ability to proliferate more readily than thymocytes from untreated tumor-bearing mice and thymocytes from untreated or melphalan-treated normal mice in response to stimulation with MOPC-315 tumor cells plus a low concentration of rIL-2. Moreover, although fresh melphalan TuB thymocytes do not contain a higher percentage of phenotypically mature cells (i.e., CD4-/CD8+ or CD4+/CD8-) than do thymocytes from normal mice or untreated tumor-bearing mice, after a 5-day culture with both MOPC-315 tumor cells and a low concentration of rIL-2, cultures of thymocytes from melphalan TuB contained a much higher percentage of CD4-/CD8+ (but not CD4+/CD8-) cells than did cultures of thymocytes from the other two sources. Since CD4-/CD8+ cells were previously shown to be responsible for the exertion of antitumor cytotoxicity by thymocytes stimulated with MOPC-315 in vitro, our results indicate that the enhanced antitumor cytotoxicity exerted by melphalan TuB thymocytes following in vitro stimulation with MOPC-315 tumor cells in the presence of a low concentration of rIL-2 is due, at least in part, to an expansion of the pool of CD4-/CD8+ effector cells.