Journal of Medical and Veterinary Mycology (1992), 30, Supplement 1, 167-177

Mechanisms of host defence against fungal infection

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R. KAPPE I, S. M. LEVITZ 2, A. CASSONE 3 AND R. G. WASHBURN 4

1Hygiene Institute, University of Heidelberg, Heidelberg, Germany; 2Section of Infectious Diseases, Evans Memorial Department of Clinical Research, University Hospital, Boston University Medical Center, Boston, Massachusetts, USA; 3Laboratory of Bacteriology and Medical Mycology, Istituto Superiore di Sanita, Rome, Italy; and 4Division of Infectious Diseases, Department of Medicine, Wake Forest University Medical Center, Winston-Salem, North Carolina, USA The prevalence of serious mycoses has dramatically increased in recent years, due in large part to the increased numbers of immunosuppressed patients, such as those with AIDS, neoplasms and transplants. An understanding of how the normal host defends against fungal invasion and what specifically goes awry in patients who develop mycoses is of fundamental importance to the development of rational approaches to immunodiagnosis and therapy of these often difficult to treat infections. Many of the exciting recent advances in our comprehension of fungal host defences have been a result of direct application of advances in basic immunology, particularly (but not exclusively) in cytokine research, definition of cell surface markers and molecular biology. A comprehensive review of all aspects of host defences against mycoses is beyond the scope of this manuscript. Rather, what follows is a selected sampling of research efforts aimed at understanding specific aspects of fungal immunology.

Natural killer (NK) cells and cytotoxic T-lymphocytes in histoplasmosis Exposure to Histoplasma capsulatum generally results in a self-limited infection in normal individuals, suggesting the existence of a remarkable host defence mechanism against the pathogen. Furthermore, it is generally accepted that the primary host defence mechanism activated in response to H. capsulatum is the cell-mediated arm of the immune response. Evidence for this concept is provided by several experimental studies: first, lymphocytes from mice immunized by sublethal infection with H. capsulatum can mediate suppression of intracellular growth of the fungus in normal mouse macrophages [19]; second, there is an increased susceptibility to histoplasmosis in congenitally athymic mice [70] or conventional mice treated with anti-lymphocyte serum and cytotoxic agents [1]; and third, anti-Histoplasma immunity can be adoptively transferred by spleen or peritoneal cells from immunized donors [22, 58, 59]. Recent work in Dr R. P. Tewari's laboratory (Springfield, Illinois) on the natural host defence against histoplasmosis has included confirmation of antifungal activity of murine circulating and peritoneal polymorphonuclear neutrophils (PMN) against H. capsulatum and enhancement of this activity by specific anti-Histoplasma Correspondence address: Stuart M. Levitz, M.D., Room E540, University Hospital, 88, E. Newton Street, Boston, MA 02118, USA. 167

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antibodies [Y. Kondoh et al., unpublished data], and demonstration of a role for NK cells in defence against murine and human histoplasmosis by providing evidence of the following: (i) significant NK cell activity in spleen and lung cells of mice (C3H/ HeN) to H. capsulatum; (ii) impaired clearance of H. capsulatum in mice depleted of NK cells; (iii) suppression of NK cell activity to H. capsulatum in mice treated with anti-asialo GM1 antibodies; (iv) increased susceptibility of mice treated with anti-asialo GM1 serum to infection with H. capsulatum [C. Raman et al., unpublished data]. Furthermore, experiments have been done to better define the role of leukocyte populations participating in the host response of immune animals to repeated challenge with H. capsulatum. Splenocytes from normal mice and mice infected subcutaneously with 104 H. capsulatum cells were studied for their cytotoxicity to H. capsulatum and YAC-1 cells. Enhanced cytotoxicity of splenocytes to both H. capsulatum and YAC-1 cells was observed from 4-7 days post-infection and the cytotoxicity returned to the control level after 21 days. Maximum antifungal activity was observed on day 7 (55.8 + 3.9 at 50:1, effector: target, vs. 29.2 + 2.8 in controls; P < 0.001) which correlated with their cytotoxicity to YAC-1 cells. The cytotoxicity was significantly increased by passage of splenocytes through nylon wool columns. The absolute number of NK cells had increased in the spleens of immune animals as indicated by increased immune spleen weights and splenocyte yields along with a constant ratio of splenocyte subpopulations in normal and immune mice as indicated by flow cytometric analysis of splenocytes. Thus, the enhanced cytotoxicity was associated with the activation of NK ceils rather than the augmentation of the relative number of NK cells. Treatment of lymphocytes from normal and immune mice (primary immunization) with anti-asialo GM1 plus complement abolished their cytotoxicity to both targets, but anti-Thy 1.2 plus complement did not. When mice were given a booster dose (10 3 H. capsulatum, intravenously (i.v.)) 3 weeks after primary infection, significant increases in both antifungal and antiYAC-1 activities were observed as early as day 1 and peaked on day 4 (P < 0.001). In contrast, the cytotoxicity of immune splenocytes was decreased by treatment with both anti-asialo GM1 serum and antiThy 1.2 plus complement. When immune splenocytes from mice given a booster dose (10 3 H. capsulatum i.v.) were depleted of Lyt 1.2 or Lyt 2.1 positive cells, both remaining lymphocyte populations had decreased anti-Histoplasma activity. These data indicate that a single sublethal infection of mice with H. capsulatum enhances their NK cell activity, and that upon repeated challenge with H. capsulatum, Tlymphocyte subpopulations are generated, which participate in antifungal activity. Taken together these experimental results point to a very early role for PMN in natural as well as in acquired immunity to histoplasmosis. NK cells play a role not only during the first week of infection, but also participate in late and chronic defence of immune individuals to further challenge with H. capsulatum. Weeks after primary contact of the host to H. capsulatum and later on, the main burden of fighting invading H. capsulatum is carried by cytotoxic T-lymphocytes along with T-helper cells.

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Inhibition and killing of Cryptococcus neoformans by stimulated human leukocyte populations Clinical and experimental studies have shown that an adequate cell-mediated immune (CMI) response is critical for effective host defences against cryptococcosis [5]. Much research has been directed at determining the specific cell phenotype(s) that is ultimately responsible for effector cell activity against C. neoformans. Activated macrophages, T-cells and NK cells, under defined conditions, have demonstrable activity against C. neoformans in in vitro murine models [11, 13, 15, 30, 45]. In vivo, the survival of mice challenged with C. neoformans has been reduced by silica treatment (which incapacitates macrophages and perhaps other effector cells) or by depletion of CD4 + T-cells (but not NK cells) [36, 42, 43). Recent reports also suggest a role for CD8 ÷ cells in pulmonary defences against cryptococcosis, perhaps by lysing unactivated macrophages laden with intracellularly replicating C. neoformans [17, 20]. Partial protection against cryptococcosis has been obtained following transfer of T-cells from immunized to naive mice or transfer of normal spleen cells (but not NKdepleted spleen cells) into cyclophosphamide-treated mice [16, 35]. These results suggest that, at least in murine models, control of cryptococcosis requires several different cell types to either act as, or to activate, anti-cryptococcal effector cells

[331. Human PMN and monocytes can kill C. neoformans in vitro, although the clinical significance of this observation is unclear since PMN and monocytes from patients with active cryptococcosis kill the fungus as well as control cells and patients with functional PMN defects rarely get cryptococcosis [9]. Nevertheless, PMN may play a role early in infection, when PMN may be the predominant inflammatory cells. However, in established infections, mononuclear cells (lymphocytes and macrophages) predominate. Thus, activation of mononuclear cells to inhibit and kill C. neoformans presumably is critical for effective host defences. Macrophages differentiate from blood monocytes that have migrated into tissues. Cells with characteristics of macrophages can be obtained following in vitro culture of blood monocytes. Such monocyte-derived macrophages have little to no activity against C. neoformans when cultured in suspension or on plastic surfaces, even if the cells are activated with interferon-7 [32]. However, since macrophage differentiation in vivo occurs while the cells are adherent to basement membrane surfaces, the ability of human monocyte-derived macrophages cultured in vitro on basement membrane components to inhibit a subsequent inoculum of C. neoformans was studied. It was found that monocytes cultured on fibronectin (but not laminin or plastic) inhibited cryptococcal growth [32]. Activity of human alveolar macrophages against C. neoformans has also been shown [68]. Moreover, in the presence of anticryptococcal antibody, non-adherent population(s) of peripheral blood mononuclear cells (PBMC), including NK cells, kill C. neoformans [6, 41]. Human lymphocytes proliferate when incubated with heat-killed C. neoformans in the presence of antigen-presenting cells, with a peak response seen following 7-9 days of culture [40]. Therefore, it was hypothesized that a consequence of this proliferative response would be the generation of effector cells capable of killing C. neoformans. PBMC were cultured with or without heat-killed C. neoformans and then challenged with an inoculum of live C. neoformans. PBMC stimulated with yeast cells (but not unstimulated PBMC) killed the encapsulated C. neoformans.

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Killing required both adherent and non-adherent cell populations, although the phenotypes of the effector cells responsible for killing await definition [33]. Recently, the capacity of the lymphocytotrophic cytokine interleukin-2 (IL-2) to activate PBMC to inhibit and kill C. neoformans was demonstrated. Optimal conditions for antifungal activity included a minimum of 5 days incubation of PBMC with IL-2, a concentration of 100 U m1-1 IL-2 and a high ratio of PBMC to fungi. Antifungal activity of IL-2 activated PBMC resided in the non-adherent fraction. Efforts are underway to determine the specific phenotype(s) of the antifungal effector cells and the requirements for their activation [26]. These studies are of particular interest because of the ability of IL-2 to activate specific populations of PBMC to become lymphokine-activated killer (LAK) cells capable of mediating non-major histocompatability complex-restricted cytotoxicity against a broad array of turnout targets both in vitro and in vivo [69]. Cell wall mannoproteins and host response to Candida albicans C. albicans is an opportunistic fungus which has become an increasingly common cause of disease in the immunocompromised host. Mucocutaneous involvement is especially common in patients with defective CMI, such as those with AIDS, whereas deep-seated candidiasis is more prevalent in neutropenic patients [18]. Although the fungus possesses an array of presumed 'virulence' factors [47], a defective immune response is usually a prerequisite for serious candidal infections to occur. As C. albicans is ordinarily a commensal micro-organism of the human GI tract, the source of disease in the immunocompromised subject is usually endogenous, and its onset is preceded by extended Candida colonization so that the multicolonized, neutropenic subject is at a great risk of developing an invasive candidiasis [47]. Thus, Candida is a micro-organism which interacts safely and, perhaps, even beneficially, in a normal host, but pathologically in a predisposed subject. In order to understand host-fungus interactions in both categories of subjects, it is important to study the fungal components which elicit, modulate or suppress host immune responses, how these components are expressed during Candida growth and morphogenesis, and the fate of these components in the host. In this context, cell wall and secretory mannoproteins are candidates to play a major role in host-Candida interaction. In fact, they are important cell surface expressed antigens capable of modulating non-Candida directed responses, mediating adhesion to host cell surfaces and binding to relevant soluble factors of immunity produced by the host. Moreover, the modulation of their cellsurface expression and secretion may be an important mechanism of immunoevasion [3, 10, 60]. Since T-cells and PMN appear to be critical for protection against mucosal and deep-seated candidiasis, respectively, studies have recently begun to identify and define the role of individual mannoproteins interacting with these cells. One mannoprotein fraction, hereafter designated MP-F2, mediated both the antigenic stimulation of human PBMC proliferation and the activation of human neutrophils. Table 1 summarizes the main immunological effects of the MP-F2 preparation. It should be stressed here that MP-F2 epitopes are variably expressed, and their secretion strongly modulated, during morphogenesis of C. albicans [60].

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TABLE 1. A synopsis of the immunogenic and immunostimulatory activities of F2 mannoprotein fraction 1. Elicitation of a strong antibody response in rabbits and mice, mostly directed against oligomannoside epitopes. In humans, it detects preexisting anti-Candida antibodies and, with high efficiency, seroconversion to high-titre anti-mannan antibodies during deep-seated candidiasis [Martino et al., personal observations]. 2. Stimulation of non-Candida, primary antibody response 'in vitro' [37]. 3. Antigen directed lymphoproliferation, cytokine production (IL-1, IL-2, IFN% TNF and IL-6) and activation of cytotoxic LAK-like effectors in human PBMC [55, 61]. 4. Activation of antimicrobial activity of human PMN 'in vitro' and production by PMN of cytokines including IL-1, TNF-ct, IL-6 [Palma et al., personal observations]. 5. Activation of murine macrophages to production of TNF-a [62].

Although MP-F2 is a substantially pure and characteristic 'mannoprotein' (mannan is > 95% of the whole polysaccharide, as detected by Fehling reagent, and mannose is essentially the only detectable sugar in HPLC analysis, unpublished data), recent experimental approaches using gradient gel SDS-PAGE, transblotting and Concanavalin A(Con A)-peroxidase, or immunodetection with anti-Candida antisera or monoclonal anti-MP antibodies, revealed the molecular complexity of this fraction. At least five polydisperse but sharply distinct molecular bands were detected by Con A-peroxidase staining. These molecules shared common oligomannoside epitopes, and their molecular mass ranged from > 200 to 34-36 kDa. The most representative molecular constituents were eluted from the gel and tested for their ability to mimic the MP-F2 fraction in stimulating the proliferation of PBMC from normal human subjects. Although still preliminary, the results have shown that the PBMC stimulatory activity is mainly associated with a molecular complex of 60--64 kDa. The stimulatory effect of the MP-F2 fraction on the antimicrobial activity of, and cytokine production by, human neutrophils was also studied. As outlined in Table 1, MP-F2 preparation is also capable of priming the antimicrobial activity of highly purified (> 99%) PMN from normal human subjects. This priming effect is seen in a range of 1-10/zg m1-1MP-F2, and is most pronounced at lower effector to target ratios. Mannoprotein stimulation of PMN anticandidal activity was studied with a large number of separate subjects, and in comparison with well known exogenous (LPS) or endogenous (GM-CSF) stimulators of PMN activity. The MP-F2 stimulatory activity was of the same order of magnitude as that of LPS and GM-CSF, and relied upon the integrity of the mannan portion of the molecule. Treatment of MPF2 with pronase (which causes the reduction or loss of the protein moiety but leaves the mannan moiety almost unaltered) abolished lymphocyte proliferation but did not affect PMN anticandidal activity. In contrast, treatment of MP-F2 with mannosidase (which degrades the mannan moiety) abolished PMN activity without affecting lymphocyte proliferation [60, 61]. In addition, these data suggest that mannan receptors may exist on the PMN surface, as they do on the monocyte surface. These findings should also be considered in the light of previous reports on the inhibitory effects of crude mannan preparations from Saccharomyces cerevisiae on PMN phagocytosis and myeloperoxidase activity.

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MP-F2 also proved as effective as LPS in the induction of message for typical monocyte cytokines as IL-1 and TNF-~t. The production of pro-inflammatory and immunomodulatory cytokines by PMN, following mannoprotein stimulation, points to a more general, previously unsuspected, immunoregulatory role of these cells, and suggests that PMN could have a part in the potent anti-tumour or anti-infection effects elicited by candidal materials in murine models [3, 55]. That activators of PMN are widespread and largely shared microbial products (e.g. LPS, mannoprotein and, probably, peptidoglycan fragments) raises the speculation that this activation serves to augment the antimicrobial activity of PMN under conditions where Fcdependent mechanisms, which require previous specific immunization, have not yet come into play. Thus, a remarkable body of experimental evidence has accumulated demonstrating that MP-F2 is a major immunogenic and immunomodulator mannoprotein complex of C. albicans, apparently with different molecular moieties coming into play in different immunological processes. Further progress in this area requires a full identification and immunochemical characterization of the various mannoprotein complexes present in the fraction, and the precise identification of receptors which, in the various cells, are expressed and, directly or indirectly, activated by the mannoprotein. Host defences against invasive Aspergillus infection

Aspergillus species are ubiquitous in the environment and inhalation exposure to the infectious conidia must be common. Yet, invasive AspergiUus infection is rare in immunologically normal hosts, a fact which attests to the existence of effective defences against the fungus. The purpose of this section is to briefly review those defences, and to describe some potential virulence factors produced by the organism. The infectious particles of Aspergillus species are inhaled conidia which are sufficiently small (mean diameter 2-3/~m) that they can reach the pulmonary alveoli. Evidence from animal models and in vitro studies indicates that the conidia become attached to bronchoalveolar macrophages [12, 21, 34, 54, 57, 64]; this initial attachment does not require complement, and Kan & Bennett presented evidence that the binding is mediated by lectin-like receptors [21]. Following attachment, conidia are ingested and efficiently killed by the macrophages, and these phagocytic cells represent the first line of host defence against invasive Aspergillus infection [34, 56, 57]. Levitz et al. showed that cationic peptides may contribute to this fungicidal activity [34]. Cortisone treatment of mice inhibits phagolysosomal fusion in bronchoalveolar macrophages and favours germination of Aspergillus conidia [39]; that experimental observation provides insight into the fact that patients who receive immunosuppressive doses of glucocorticoids are at risk for invasive pulmonary aspergillosis. In addition to bronchoalveolar macrophages, peripheral blood phagocytes contribute toward host defence against the infection and patients with leukopenia or chronic granulomatous disease are at an increased risk for invasive aspergillosis [4, 14]. Optimal phagocytosis of Aspergillus conidia by peripheral blood phagocytic cells is possible only after the fungal particles have been opsonized with serum [25, 66, 67]. Conidia are known to activate the alternative complement pathway, resulting in opsonization of the target particles with complement component C3 (in the forms C3b and iC3b; [24]) and generation of the chemoattractant C5a [63]. Polymorphonuclear

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neutrophils ingest and kill swollen conidia by oxidative myeloperoxidase - or ferrous ion-dependent mechanisms and via the defensins [27, 29, 34]. In contrast, resting conidia are relatively resistant to the fungicidal effect of neutrophils; these fungal particles are only inefficient stimulators of the neutrophil respiratory burst and degranulation and they are resistant to neutrophil oxidants and defensins [25, 29, 31]. However, mononuclear phagocytes do exert fungicidal activity against resting conidia [56, 66], and there is evidence that peripheral blood monocytes utilize oxidative myeloperoxidase-dependent and -independent pathways to kill the conidia [66]. Conidia which escape from early host defences germinate into hyphae, the elongated structures which represent the tissue-invasive form of Aspergillus species. These septate, approximately 2 /.Lm-wide, dichotomously branching structures account for most of the clinical manifestations of invasive aspergillosis because they invade blood vessels, producing distal thrombosis and tissue necrosis. Peripheral blood neutrophils and monocytes can attach to hyphae in the absence of complement and they are able to damage the fungi as judged by electron microscopic, isotopic and staining criteria using the tetrazolium salt, MTT (3-[4,5 dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) [7, 8, 28, 50]. Diamond et al. provided evidence that neutrophils damage Aspergillus hyphae by oxidative mechanisms which could be inhibited by scavengers of hydrogen peroxide or hydroxyl radicals, and that monocytes also damage hyphae by a myeloperoxidase-dependent oxidative process [7]. Recently, Rex and coworkers confirmed that the myeloperoxidase system contributes to neutrophil-mediated hyphal damage, using the MTT assay [50]. Neutrophils from patients with chronic granulomatous disease exhibit poor baseline ability to damage Aspergillus hyphae in vitro. However, the damage can be augmented by in vivo treatment with recombinant interferon-% a finding which provides hope that the agent will be useful for prevention and/or treatment of invasive aspergillosis in that group of compromised patients [49]. It is logical to postulate that peripheral blood phagocytes would need to follow a gradient of the chemoattractant C5a toward foci of hyphal invasion. Therefore, a fungal inhibitor of alternative complement pathway activation could potentially serve as a virulence factor. Aspergillus fumigatus hyphae are known to produce such an inhibitor in vitro [65, 67]. The inhibitor, which contains polar lipids, may function to the advantage of the invading fungus by impairing generation of C5a in vivo. Studies from our laboratory show that the complement inhibitor is produced more efficiently by the pathogenic species A. fumigatus and Aspergillus flavus than by the relatively non-pathogenic species AspergiUus niger, a finding which further enhances the possibility that the inhibitor may serve as a virulence factor. In addition, Aspergillus species produce inhibitors of phagocytosis, including aflatoxin and gliotoxin [44, 53], and there is evidence that the enzyme elastase may contribute to the virulence of Aspergillus. This metalloproteinase is found more commonly in isolates of Aspergillus from patients with invasive disease than those with colonization [51, 52], and animal model data suggest that elastase production correlates with tissue invasion [23]. A number of additional Aspergillus proteases, including collagenase could also potentially contribute to the pathogenesis of invasive aspergillosis [38, 46]. In summary, normal hosts possess effector cells including alveolar macrophages, polymorphonuclear neutrophils and monocytes which defend against invasive aspergillosis by killing conidia and damaging hyphae. The alternative complement pathway probably contributes toward protection by generating the chemoattractant C5a and

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by opsonizing inhaled conidia for ingestion and killing by peripheral blood phagocytic cells. These defences may be counterbalanced by fungal virulence factors, including elastase, inhibitors of phagocytosis such as aflatoxin and gliotoxin, and a lipidcontaining inhibitor of alternative complement pathway activation. Thus, clinical outcome of invasive Aspergillus infection is probably determined by competition between phagocytic host defences and fungal virulence factors. In summary, as the above examples illustrate, a complex interplay exists in the host between fungal virulence factors favouring disease, and immune and non-immune host mechanisms defending against disease. Moreover, the host defences necessary to prevent mycoses are unique for each fungus. Thus, while neutrophils are essential in the defence against aspergillosis and deep-seated candidiasis, CMI appears of paramount importance in defence against histoplasmosis, cryptococcosis and mucocutaneous candidiasis. Fortunately, in the immunologically intact individual, the host nearly always comes out the winner. (Although, it could be argued that the host wins the battle but the fungus wins the war, since, upon the death of the host, fungi are the eventual winners.) However, in the immunologically impaired host, the balance is often tipped in favour of the fungus, and serious mycoses can result. CONTRIBUTORS The contributors to this symposium were: R. Kappe, Natural killer cells and cytotoxic T-lymphocytes in histoplasmosis; S. M. Levitz, Inhibition and killing of C. neoformans by stimulated human leukocyte populations; A. Cassone, Cell wall mannoproteins and host response to C. albicans; R. Washburn, Host defences against invasive Aspergillus infection. The co-convenors were S. M. Levitz and R. Kappe. REFERENCES 1. ADAMSON,D. M. & COZAD,G. C. 1969. Effect of antilymphocyte serum on animals experimentally infected with Histoplasma capsulatum or Cryptococcus neoformans. Journal of Bacteriology, 100, 1271-1276. 2. AUSlELLO,C. M., PALMA,C., SPAGNOLI,G. C., PIAZZA,A., CASCIAN1,C. U. • CASSONE,A. 1989. Cytotoxic effectors in human peripheral blood mononuclear cells induced by a mannoprotein complex of Candida albicans: a comparison with interleukin-2 activated killer cells. Cellular Immunology, 121, 349-359. 3. CASSONE,A., MARCONI,P., BISTONI,F., MATTIA,E., SBARAGLIA,G., GARACI,E. & BONMASSAR,E. 1981. Immunoadjuvant effects of Candida albicans and its cell wall fractions in a mouse lymphoma model. Cancer Immunology and Immunotherapy, 10, 181-190. 4. COHEN, M. S., ISTURIZ, R. E., MALECH, H. L., ROOT, R. K., WILFERT, C. M., GUTMAN, L. & BUCKLEY, I"I. R. 1981. Fungal infection in chronic granulomatous disease. The importance of the phagocyte in defense against fungi. American Journal of Medicine, 71, 59-66. 5. DIAMOND,R. n. 1990. Cryptococcus neoformans. In: G. L. MANDELL,R. G. DOUGLAS,JR, & J. E. BENNETT (Eds) Principles and Practice of Infectious Diseases, 3rd edn., pp. 1980-1989. Churchill Livingstone, New York. 6. DIAMOND, R. D. & ALLISON, A. C. 1976. Nature of the effector cells responsible for antibodydependent cell-mediated killing of Cryptococcus neoformans. Infection and Immunity, 14, 716--720. 7. DIAMOND,R. D., HUBER,E. & HAUDENSCHILD,C. C. 1983. Mechanisms of destruction of Aspergillus fumigatus hyphae mediated by human monocytes. Journal of Infectious Diseases, 147, 474--483. 8. DIAMOND,R. D., KRZESlCKI,R., EPSTEIN, B. & JAO,W. 1978. Damage to hyphal forms of fungi by

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Infection and Immunity, 51,547-555. 17. HILL, J. O. & HARMSEN, A. G. 1991. Intrapulmonary growth and dissemination of Cryptococcus neoformans in mice depleted of CD4+ or CD8+ T-cells. Journal of Experimental Medicine, 173, 755-758. 18. HORN, R., WONG, B., KIEHN, T. E. & ARMSTRONG,D. 1985. Fungemia in a cancer hospital: changing frequency, earlier onset, and results of therapy. Reviews of Infectious Diseases, 7, 646--655. 19. HOWARD,D. H. & OTTO, V. 1977. Experiments of lymphocyte-mediated cellular immunity in murine histoplasmosis. Infection and Immunity, 16, 226-231. 20. HUFFNAGLE,G. B., YATES, J. L. & LlPSCOMB,M. F. 1991. Immunity to a pulmonary Cryptococcus neoformans infection requires both CD4+ and CD8+ T-cells. Journal of Experimental Medicine, 173, 793-800. 21. KAN, V. L. & BENNETT, J. E. 1988. Lectin-like attachment sites on murine pulmonary alveolar macrophages bind Aspergillus fumigatus conidia. Journal of Infectious Diseases, 158, 407-414. 22. KHARDOPd,N., CHAUDnARV,S., MCCONNACmE,P. & TEWARI,R. P. 1983. Characterization of lymphocytes responsible for protective immunity to histoplasmosis in mice. Mykosen, 26, 523-532. 23. KOTHARY,M. H., CHASE, T., JR. & MACMILLAN,J. D. 1984. Correlation of elastase production by some strains of AspergiUus fumigatus with ability to cause pulmonary invasive aspergillosis in mice. Infection and Immunity, 43, 320-325. 24. KOZEL, T. R., WILSON, M. A., FARRELL,T. P. ¢~ LEVITZ, S. M. 1989. Activation of C3 and binding to Aspergillus fumigatus conidia and hyphae. Infection and Immunity, 57, 3412-3417. 25. LEHRER,R. I. & JAN, R. G. 1970. Interaction of Aspergillus fumigatus spores with human leukocytes and serum. Infection and Immunity, 1,345-350. 26. LEVITE, S. M. 1991. Activation of human peripheral blood mononuclear cells by interleukin-2 and granulocyte-macrophage colony stimulating factor to inhibit Cryptococcus neoformans. Infection and Immunity, 59, 3393-3397. 27. LEVITZ,S. M. & DIAMOND,R. D. 1984. Killing of Aspergillus fumigatus spores and Candida albicans yeast phase by the iron-hydrogen peroxide-iodide cytotoxic system: comparison with the myeloperoxidase-hydrogen peroxide-halide system. Infection and Immunity, 43, 1t00-1102. 28. LEVlTZ, S. M. & DIAMOND, R. D. 1985. A rapid colorimetric assay of fungal viability with the tetrazolium salt MTT. Journal of Infectious Diseases, 152, 938-945. 29. LEVlTZ,S. M. & DIAMOND,R. D. 1985. Mechanisms of resistance of Aspergillus fumigatus conidia to killing by neutrophils in vitro. Journal of Infectious Diseases, 152, 33-42. 30. LEVlTZ,S. M. & DIBENEDETTO,D. J. 1988. Differential stimulation of murine resident peritoneal cells by selectively opsonized encapsulated and acapsular Cryptococcus neoformans. Infection and Immunity, 56, 2544-2551. 31. LEvrrz, S. M. & FARRELL,T. P. 1990. Human neutrophil degranulation stimulated by Aspergillus fumigatus. Journal of Leukocyte Biology, 47, 170-175.

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