Lymphocytes constitute a critical component of host defenses against cryptococcosis. Previously, we demonstrated that human lymphocytes cultured with interleukin-2 formed conjugates with, and directly inhibited the growth of, Cryptococcus neoformans. Here, we explore the anticryptococcal activity of freshly isolated, highly purified populations of human peripheral blood lymphocytes. Lymphocytes were incubated with encapsulated C. neoformans for 24 h, after which the lymphocytes were lysed, dilutions and spread plates were made, and CFU were counted. Fungistasis was determined by comparing growth in wells with and without lymphocytes. Nylon wool-nonadherent peripheral blood mononuclear cells (NWNA PBMC) were highly fungistatic, even if either T cells or natural killer (NK) cells were depleted by panning. A mixed population of T cells and NK cells, obtained by rosetting NWNA PBMC with sheep erythrocytes, completely inhibited cryptococcal growth, whereas the nonrosetting cells had little fungistatic activity. CD4+, CD8+, and CD16/56+ lymphocytes, isolated by positive immunoselection, had potent growth-inhibitory activity. In contrast, purified B cells had no activity. Fungistasis was seen even in the absence of opsonins. Antifungal activity was markedly diminished when surface receptors on NWNA PBMC were cleaved by treatment with trypsin or bromelain. Supernatants from stimulated lymphocytes or concentrated lymphocyte sonicates were not active. Lymphocyte-mediated fungistasis was seen with two different strains of C. neoformans. CD4+, CD8+, and CD16/56+ lymphocytes formed conjugates with C. neoformans, as observed under Nomarski differential interference contrast microscopy and videomicroscopy. These data demonstrate that freshly isolated peripheral blood T cells and NK cells have the capacity to bind and directly inhibit the growth of C. neoformans.

Candida albicans absorbed with yeast-phase organisms preferentially stained germ tube segments of several strains of mycelial-phase C. albicans by the indirect fluorescent-antibody staining technique. Germ tube segment antigens were not found in significant amounts on blastospore segments or on yeast-phase organisms. Absorption of the mycelial-phase reference sera with yeastphase C. stellatoidea, but not with C. tropicalis, C. guillermondii, or Saccharomyces cerevesiae, resulted in preferential germ tube segment staining of C. albicans. A dithiothreitol extract of mycelial-phase C. albicans organisms blocked staining of the germ tube segment, but a dithiothreitol extract of yeast-phase organisms did not. When dithiothreitol extracts from both phases were reacted against yeast-absorbed reference sera in tandem crossed and crossed line immunoelectrophoresis, a cross-reacting arc and several arcs unique to the mycelial-phase extract were noted. Immunofluorescent staining tests were performed, using appropriately absorbed sera from patients with candidiasis to stain a laboratory strain of C. albicans. Human tissue slices infected with C. albicans were used as targets for appropriately absorbed rabbit antisera. These human data indicated that antigens preferentially expressed on the germ tube in vitro were also expressed on filamentous structures of the fungus in infected human tissues. In vitro and in vivo, the invasive mycelial phase of C. albicans expresses certain antigens that are highly concentrated on the germ tube. Infections by Candida albicans and other species of Candida occur frequently in imnmunocompromised patients (32). Manifestations of visceral candidiasis are frequently subtle and blood cultures are often negative (40). When blood cultures are positive, distinguishing transient fungemia (such as that seen with intravenous catheters) from systemic disease can be difficult (9). To provide a noninvasive aid to diagnosis, many serological tests have been developed, but as it is difficult to grow large quantities of mycelial-phase C. albicans organisms (29), nearly all serological tests have detected antibodies against antigens isolated from yeastphase organisms or antigens present in large amounts in

Several clinical and laboratory isolates of Candida albicans have a natural blue surface fluorescence when cultured and observed with sensitive optics. The localization and color of the fluorescence are similar to those of the natural fluorescence of sporulated Saccharomyces cerevisiae which is caused by the generation and surface deposition of the cross-linking amino acid dityrosine. In S. cerevisiae, dityrosine production results from the direct action of at least two genes and is responsible for resistance of the ascospores to lytic enzymes and physicochemical trauma. Among the criteria for the identification of dityrosine is pH sensitivity of the fluorescence intensity and a highly characteristic shift of the fluorescence excitation maximum with a change in pH. Video microscopy of whole Candida organisms revealed the characteristic dityrosine intensity maximum at pH ϳ10 and the intensity minimum at pH ϳ2. Separation of an acid hydrolysate of Candida cell walls by reverse-phase high-performance liquid chromatography revealed a fluorescence peak that coelutes with the reagent dityrosine. At pH ϳ10, this peak has a fluorescence excitation maximum of 320 to 325 nm, while at pH ϳ2, the excitation maximum is 285 to 290 nm. This excitation maximum shift and the observed emission maximum of ϳ410 nm are characteristic of dityrosine. Two separate strains of C. albicans were injected intraperitoneally into mice and harvested at 24 h. Blue surface fluorescence was observed, suggesting that dityrosine generation occurs in vivo as well as in vitro. This is the first report of the presence of dityrosine in a human fungal pathogen. Candida albicans and several other Candida species are major pathogens in hospitalized, immunocompromised patients (4). The mechanisms allowing C. albicans to colonize mucosal surfaces and then invade the bloodstream and visceral organs have long been under intense investigation (32), with a large number of putative virulence factors proposed as contributing to pathogenicity (15). Studies of the Candida cell surface (36) have been of particular interest because of its importance in antigenicity (11, 39), in adherence to epithelial and endothelial cells (11), and in interactions with the host immune system (24, 38, 40, 41) and because the mechanisms of cell wall synthesis are critical for our understanding of morphogenesis and for development of new antifungal agents (25). The Candida cell wall is composed principally of mannan (linked with protein to form mannoprotein), ␣-glucan, ␤-glucan, and chitin, which are complex polymers of mannose, glucose, and N-acetylglucosamine, respectively. The content and distribution of these components vary with cell age, culture conditions, and morphology (25, 36). The linkages between the glucan, chitin, and mannoprotein are poorly understood but presumably play a critical role in maintaining the integrity of the organism. Dityrosine, a relatively rare amino acid, is well known as a crosslinking agent that is essential for the resistance of several species, inclu...