Experimental Cryptococcosis Research Articles

Iron Regulation of the Major Virulence Factors in the AIDS-Associated Pathogen Cryptococcus neoformans

Iron overload is known to exacerbate many infectious diseases, and conversely, iron withholding is an important defense strategy for mammalian hosts. Iron is a critical cue for Cryptococcus neoformans because the fungus senses iron to regulate elaboration of the polysaccharide capsule that is the major virulence factor during infection. Excess iron exacerbates experimental cryptococcosis and the prevalence of this disease in Sub-Saharan Africa has been associated with nutritional and genetic aspects of iron loading in the background of the HIV/AIDS epidemic. We demonstrate that the iron-responsive transcription factor Cir1 in Cr. neoformans controls the regulon of genes for iron acquisition such that cir1 mutants are “blind” to changes in external iron levels. Cir1 also controls the known major virulence factors of the pathogen including the capsule, the formation of the anti-oxidant melanin in the cell wall, and the ability to grow at host body temperature. Thus, the fungus is remarkably tuned to perceive iron as part of the disease process, as confirmed by the avirulence of the cir1 mutant; this characteristic of the pathogen may provide opportunities for antifungal treatment.

An Innate Immune System Cell Is a Major Determinant of Species-Related Susceptibility Differences to Fungal Pneumonia

Rats and mice are considered resistant and susceptible hosts, respectively, for experimental cryptococcosis. For both species, alveolar macrophages (AM) are central components of the host response to pulmonary Cryptococcus neoformans infection. We explored the role of AM in three strains of mice and three strains of rats during cryptococcal infection by comparing the outcome of infection after macrophage depletion using liposomal clodronate. AM depletion was associated with enhancement and amelioration of disease in rats and mice, respectively, as measured by lung fungal burden. The apparent protective role for AM in rats correlated with enhanced anti-cryptococcal activity as measured by phagocytic activity, oxidative burst, lysozyme secretion, and ability to limit intracellular growth of C. neoformans. Furthermore, rat AM were more resistant to lysis in association with intracellular infection. In summary, differences in AM function in rats and mice suggest an explanation for the species differences in susceptibility to C. neoformans based on the inherent efficacy of a central effector cell of the innate immune system.

MyD88 and TLR2, but not TLR4, are required for host defense against Cryptococcus neoformans

We investigated here the potential role of Toll-like receptors (TLR) and the adaptor protein MyD88 in innate immunity responses to Cryptococcus neoformans, a pathogenic encapsulated yeast. Peritoneal macrophages from MyD88(-/-) or TLR2(-/-) mice released significantly less TNF-alpha, compared with wild-type controls, after in vitro stimulation with whole yeasts. In contrast, no differences in TNF-alpha release were noted between macrophages from C3H/HeJ mice, which have a loss of function mutation in TLR4, relative to C3H/HeN controls. When MyD88- or TLR2-deficient mice were infected with low doses of the H99 serotype A strain, all of the control animals, but none of MyD88(-/-) and only 38% of the TLR2(-/-) animals survived, in association with higher fungal burden in the mutant mice. Both MyD88(-/-) and TLR2(-/-) animals showed decreased TNF-alpha, IL-12p40 and/or IFN-gamma expression in various organs during infection. No difference in susceptibility to experimental cryptococcosis was found between C3H/HeJ mice and C3H/HeN controls. In conclusion, our data indicate that TLR2 and MyD88, but not TLR4, critically contribute to anti-cryptococcal defenses through the induction of increased TNF-alpha, IL-12 and IFN-gamma expression.

Mechanisms for Induction of Immunosuppression during Experimental Cryptococcosis: Role of Glucuronoxylomannan

In previous work we have demonstrated that spleen mononuclear (Spm) cells from rats obtained 14 days after infection with Cryptococcus neoformans showed a diminution in proliferative response to Concanavalin A (Con A). In this study we further investigate some characteristics of the Spm cell population involved in the immunosuppressor phenomenon induced by C. neoformans. We observed that unstimulated Spm cells expressing T-cell receptor (TCR+) from infected rats were reduced in number after 96 h of culture. When the Spm cells from infected rats were stimulated with Con A, increased production of IL-10, reduced levels of IL-2, and decreased CD11a surface expression were shown. These immunosuppressor phenomena were also observed when the capsular polysaccharide, glucuronoxylomannan (GXM), was added to cultures of Spm cells from normal rats. However, GXM had a more pronounced effect in reducing the number of cells surviving in culture than that observed during infection and produced an increase in IL-4 production by Con-A-stimulated Spm cells. Addition of anti-IL-10 monoclonal antibody to cultures restored the lymphoproliferation of Spm cells from infected animals, indicating that IL-10 production is a suppressor mechanism of cell-mediated immunity during experimental infection. The results presented here indicate that at least two mechanisms mediate the nonspecific suppression in this model of cryptococcosis: IL-10 production and diminution of the number of T cells. GXM could be involved, since it has a pronounced effect in the reduction of Spm cells in vitro.

A cryptococcal capsular polysaccharide mimotope prolongs the survival of mice with Cryptococcus neoformans infection.

Defined Abs to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) have been shown to be protective against experimental cryptococcosis. This suggests that if a vaccine could induce similar Abs it might protect against infection. However, the potential use of a GXM-based vaccine has been limited by evidence that GXM is a poor immunogen that can induce nonprotective and deleterious, as well as protective, Abs, and that the nature of GXM oligosaccharide epitopes that can elicit a protective response is unknown. In this study, we investigated whether a peptide surrogate for a GXM epitope could induce an Ab response to GXM in mice. The immunogenicity of peptide-protein conjugates produced by linking a peptide mimetic of GXM, P13, to either BSA, P13-BSA, or tetanus toxoid, P13-tetanus toxoid, was examined in BALB/c and CBA/n mice that received four s.c. injections of the conjugates at 14- to 30-day intervals. All mice immunized with conjugate produced IgM and IgG to P13 and GXM. Challenge of conjugate-immunized mice with C. neoformans revealed longer survival and lower serum GXM levels than control mice. These results indicate that 1) P13 is a GXM mimotope and 2) that it induced a protective response against C. neoformans in mice. P13 is the first reported mimotope of a C. neoformans Ag. Therefore, the P13 conjugates are vaccine candidates for C. neoformans and their efficacy in this study suggests that peptide mimotopes selected by protective Abs deserve further consideration as vaccine candidates for encapsulated pathogens.

Urease as a virulence factor in experimental cryptococcosis.

Urease catalyzes the hydrolysis of urea to ammonia and carbamate and has been found to be an important pathogenic factor for certain bacteria. Cryptococcus neoformans is a significant human pathogenic fungus that produces large amounts of urease; thus we wanted to investigate the importance of urease in the pathogenesis of cryptococcosis. We cloned and sequenced the genomic locus containing the single-copy C. neoformans urease gene (URE1) and used this to disrupt the native URE1 in the serotype A strain H99. The ure1 mutant strains were found to have in vitro growth characteristics, phenoloxidase activity, and capsule size similar to those of the wild type. Comparison of a ure1 mutant with H99 after intracisternal inoculation into corticosteroid-treated rabbits revealed no significant differences in colony counts recovered from the cerebrospinal fluid. However, when these two strains were compared in both the murine intravenous and inhalational infection models, there were significant differences in survival. Mice infected with a ure1 strain lived longer than mice infected with H99 in both models. The ure1 strain was restored to urease positivity by complementation with URE1, and two resulting transformants were significantly more pathogenic than the ure1 strain. Our results suggest that urease activity is involved in the pathogenesis of cryptococcosis but that the importance may be species and/or infection site specific.

Fluconazole, with or without dexamethasone for experimental cryptococcosis: impact of treatment timing.

The time of initiation of fluconazole treatment with or without dexamethasone, and the impact on mycological outcome and drug pharmacokinetics were assessed in a murine model of disseminated cryptococcosis. Non-infected mice and mice with disseminated cryptococcosis were given saline, dexamethasone, or fluconazole +/- dexamethasone, 1 or 8 days after infection. Cfus were counted in tissues, and fluconazole concentrations were determined in plasma and tissues by HPLC and a bioassay. Despite fluconazole tissue and plasma concentrations which were above the minimal inhibitory concentration, the numbers of cfus in brain and lung tissues were reduced after early (P = 0.002 and 0.04, respectively), but not after late fluconazole treatment. The administration of dexamethasone did not have a deleterious effect on the number of cfus, fluconazole pharmacokinetics or antifungal activity. In conclusion, the size of the fungal burden influences the effective level of fluconazole activity in lung and brain. These results strongly suggest that potential antifungal agents should be studied following both early and late administration in experimental cryptococcosis.

Involvement of nitric oxide in protecting mechanism during experimental cryptococcosis.

In the present study we investigated the role of nitric oxide (NO) in the effector mechanisms of host defense against Cryptococcus neoformans in vivo. Our results showed an increase of NO produced by the peritoneal macrophages from 14-days infected rats compared with normal rats. These cells were capable of killing C. neoformans to a greater extent than macrophages from noninfected rats (80% vs 20%, respectively). The killing of C. neoformans by infected cells was efficiently inhibited (80% to 35%, P < 0.001) by adding aminoguanidine (AG) to the cultures. We observed that in vivo administration of AG to the infected animals efficiently inhibited the metabolism producing NO and failed to affect that of normal animals. When the NO synthase (NOS) was inhibited in vivo in the infected animals, a marked increase of the fungi charge in the organs was observed with respect to the normal animals treated with AG. We also observed that the course of the infection is drastically modified after the inhibition of NO production because all the animals infected and treated with AG died from cryptococcosis before 20 days postinfection (p.i.). These results indicate that NO is a crucial molecule in the effector mechanisms in this infection model.

Fungemia during murine cryptococcosis sheds some light on pathophysiology

We studied fungemia over time in outbred mice infected with Cryptococcus neoformans and looked at its relationship with the intravenous (i.v.) inoculum size, tissue burden and survival. Fungemia was evaluated by culture of 10 microl of peripheral blood from living mice or by culture of buffy coats from sacrificed animals. For all inoculum sizes studied, fungemia could last several weeks after the i.v. inoculation. Individual susceptibility of outbred mice to cryptococcal infection was evidenced by variations in the course, duration and magnitude of fungemia and tissue localizations. These results suggest that the fungus can recirculate after the initial i.v. inoculation. Fungemia, assessed by culture of buffy coats, correlated with the extent of infection in the spleen, lung or brain (P<<0.001) on day 1 after inoculation but only with yeast burden in lung or spleen on day 8, thus demonstrating that brain reacts differently to C. neoformans infection than other organs. Comparison of blood culture techniques and examination of smears suggest that cryptococci might circulate within leucocytes. Finally, quantitative blood cultures may accurately assess the fungal load during experimental cryptococcosis.

Lack of Involvement of Nitric Oxide in the Macrophage-Mediated Inhibition of Spleen Cell Proliferation during Experimental Cryptococcosis

We investigated the proliferative response to mitogens of spleen mononuclear (Spm) cells fromCryptococcus neoformans-infected rats. We determined reactive oxygen intermediates (ROI) and nitric oxide (NO) production by peritoneal and Spm cells, and evaluated the correlation of the proliferative response with NO and ROI production. The proliferative response of Spm cells from infected rats dramatically decreased at 14 and 21 days postinfection (PI). The unresponsiveness of Spm cells from 14-day infected rats was not abrogated by the addition of L-NAME and AG, indicating that NO is not involved in the antiproliferative response of experimental cells. When SOD, catalase, and indomethacin were added to the cultures, the suppression was still observed, indicating that ROI and prostaglandins are not involved in the unresponsiveness of lymphocytes. The proliferative response of lymphocytes from 14-day infected rats was significantly improved when cultures were made in the presence of Con A and exogenous IL-2. Additionally, a purified T-rich fraction from infected rats cultured with control macrophages recovered the normal proliferative response. This result indicates that macrophages from infected rats mediate the unresponsiveness of lymphocytes, probably by reducing the ability of lymphocytes to secrete IL-2.

Chloroquine induces human mononuclear phagocytes to inhibit and kill Cryptococcus neoformans by a mechanism independent of iron deprivation.

Infections due to Cryptococcus neoformans are common in AIDS patients. We investigated the effect of chloroquine, which raises the pH of phagolysosomes, on the anticryptococcal activity of mononuclear phagocytes. C. neoformans multiplied within monocyte-derived macrophages (MDM) in the absence of chloroquine but were killed with the addition of chloroquine. Ammonium chloride was also beneficial, suggesting that effects were mediated by alkalinizing the phagolysosome. Chloroquine inhibits growth of other intracellular pathogens by limiting iron availability. However, chloroquine-induced augmentation of MDM anticryptococcal activity was unaffected by iron nitriloacetate, demonstrating that chloroquine worked by a mechanism independent of iron deprivation. There was an inverse correlation between growth of C. neoformans in cell-free media and pH, suggesting that some of the effect of chloroquine on the anticryptococcal activity of MDM could be explained by relatively poor growth at higher pH. Chloroquine enhanced MDM anticryptococcal activity against all tested cryptococcal strains except for one large-capsule strain which was not phagocytosed. Positive effects of chloroquine were also seen in monocytes from both HIV-infected and -uninfected donors. Finally, chloroquine was therapeutic in experimental cryptococcosis in outbred and severe combined immunodeficient mice. Thus, chloroquine enhances the activity of mononuclear phagocytes against C. neoformans by iron-independent, pH-dependent mechanisms and is therapeutic in murine models of cryptococcosis. Chloroquine might have clinical utility for the prophylaxis and treatment of human cryptococcosis.

Immunosuppression in experimental cryptococcosis: Variation of splenic and thymic populations and expression of class II major histocompatibility complex gene products

Previous studies from our laboratory have shown that infection with Cryptococcus neoformans can trigger the production of a series of suppressor cells that specifically inhibit the cell-mediated immune response to a nonrelated antigen, the human serum albumin (HSA). In the present study, we determined the variation of thymus and spleen cell populations in rats infected with C. neoformans and immunized with HSA-CFA at the time when suppressor activity was demonstrated. At 21 days postinfection, the number and the percentage of CD4 +CD8 + cells were significantly increased in the thymus together with a minor imbalance in other thymocytes subsets. The study of two class II molecules encoded within the major histocompatibility complex, IA and IE, showed that the total number of class II IA-positive cells was increased in the glands of animals infected when compared to the glands of animals only immunized, while the corresponding percentages were lower than those in control rats. On the contrary a significant increase in both the number and the percentage of IE phenotype was observed in the thymus of infected rats, compared to the animals that were only immunized and used as a control. The IE/IA phenotype ratio within each group was increased in rats injected with the fungus. The study of spleen populations revealed an increase in CD4 + and CD8 + cells and a decrease in the B cells. The IE antigen was increased in the spleen of infected animals. The IA molecule expression showed no difference between the infected animals and the control groups. The IE/IA phenotypes ratio was mildly increased in the spleen of infected rats. These findings reveal that the cryptococcal infection can render an important imbalance in the thymus and spleen T-cell compartment together with a significant increase in the expression of the EE molecule at the time when the suppressor activity was demonstrated in this model.

Cytokine treatment of central nervous system infection: efficacy of interleukin-12 alone and synergy with conventional antifungal therapy in experimental cryptococcosis.

Cell-mediated immune responses appear to be critical in the outcome of cryptococcosis. Interleukin-12 (IL-12) was studied for its potential use as a therapeutic agent because of its stimulation of natural killer cells and gamma interferon production by stimulated T cells and natural killer cells. Gamma interferon-activated macrophages are important in host resistance against cryptococcosis. In two separate studies, male BALB/c mice were infected intravenously with Cryptococcus neoformans. In the first study, mice received either no treatment, 5.0 mg of fluconazole alone per kg of body weight per day (by gavage twice daily), or IL-12 subcutaneously at 0.01, 0.1, or 1.0 microgram/day once daily (low-dose study) alone or in combination with 5.0 mg of fluconazole per kg/day. In a second study (high dose), the dosages of IL-12 used were 1.0, 2.5, or 5.0 micrograms/day. Therapy was given for 10 consecutive days, and the number of CFU of C. neoformans remaining in various organs was quantitated 1 or 2 days after administration of the last dose. In the low-dose study, IL-12 at 0.1 or 1.0 microgram reduced the level of brain infection by approximately 10-fold (P < 0.05) and IL-12 at 1.0 or 0.1 microgram/day enhanced the efficacy of fluconazole. In liver, both the efficacy of IL-12 alone (0.01 or 0.1 microgram; P < 0.05) and enhancement of the efficacy of fluconazole (P < 0.05) were seen. No efficacy of IL-12 was seen in spleens or lungs, although spleen weights increased fourfold in mice given 1.0 microgram of IL-12 per day. In the high-dose study, all IL-12 doses alone again reduced the levels of brain infection (5- to 8-fold; P < 0.05) when the two were given in combination. No overt toxicities were observed at any dose, and overall, 1.0 microgram of IL-12 per day was found to be the optimal dosage for reducing infection in the brain. To our knowledge, this is the first demonstration of the efficacy of cytokine therapy in systemic and particularly brain infections with C. neoformans. The stimulation of cell-mediated immunity represents a new approach to therapy and can enhance suboptimal antimicrobial chemotherapy. IL-12 should be considered for further study and for clinical trials. These studies suggest that other opportunistic central nervous system pathogens should also be investigated.

Tissue distribution and antifungal effect of liposomal itraconazole in experimental cryptococcosis and pulmonary aspergillosis.

We studied the tissue distribution and in vivo antifungal effect of itraconazole, incorporated into pure dipalmitoylphosphatidylcholine (DPPC) multilamellar liposomes and administered intravenously. Eighty percent of the itraconazole was associated with DPPC. Drug levels in lung, brain, and liver, obtained after intravenous administration of tritiated itraconazole, were higher when the drug was administered intravenously as liposomal than when it was dissolved in cyclodextrin. Administration of the liposomal formulation also led to higher and sustained levels of intact itraconazole in serum. Efficacy was assessed in DBA/2 mice infected intravenously with 3 x 10(6) Cryptococcus neoformans, an inoculum responsible for early fatal pneumonia, or 3 x 10(5) C. neoformans, leading to delayed meningitis. In pneumonia, 20 mg/kg of liposomal itraconazole was more effective on survival than the same dose given intravenously in cyclodextrin or twice the dose administered orally dissolved in polyethylene glycol 200. In meningitis, liposomal itraconazole was also more efficient than the drug dissolved in cyclodextrin. These results were confirmed by colony counts in the brain and lung of infected mice. In immunosuppressed OF1 mice infected after inhalation of Aspergillus fumigatus spores, liposomal itraconazole (20 mg/kg x 3) was the only effective treatment. We conclude that intravenous liposomal delivery of itraconazole enhances both concentrations in infected tissues and the in vivo efficacy of the drug. Such passive targeting of antifungal agents other than amphotericin B might be helpful in the treatment of severe systemic mycoses, especially in the case of lung or brain involvement.

Treatment of experimental cryptococcosis with SCH 39304 and fluconazole

The efficacy of two triazoles, SCH 39304 and fluconazole, in the treatment of disseminated cryptococcosis in Wistar rats was determined. A total of 160 rats were inoculated intracardiacally with 2 x 10(5) cells of Cryptococcus neoformans. Both drugs were administered by gavage once daily, at three doses (8, 16, and 32 mg/kg/day). Two treatment schedules were followed: (i) treatment began 1 week after infection and continued for 3 weeks and (ii) prophylaxis treatment began 3 days before infection and continued an additional 3 weeks. Evaluation was based on (i) macroscopic examination of lungs, (ii) microscopic examination of brains and lungs, (iii) histopathology of brains and lungs, and (iv) determination of number of CFU in brains. The number of CFU was the best measure of activity. SCH 39304 was more active than fluconazole in both regimens, and, prophylactically, SCH 39304 was able to achieve biological cures.

Improved Amphotericin B Activity by a Monoclonal Anti-Cryptococcus neoformans Antibody: Study during Murine Cryptococcosis and Mechanisms of Action

Current therapy of cryptococcosis is unsatisfactory, particularly in patients with AIDS. Experimental cryptococcosis models in DBA/2 mice were used to determine whether the murine monoclonal anticryptococcal antibody (designated E1) might potentiate the chemotherapeutic effect of amphotericin B (AmB). According to the inoculum size, these mice died spontaneously from acute pneumonia (high inoculum) or from brain swelling (lower inoculum). AmB and E1 together significantly improved the survival of mice in both models compared with AmB alone. The mechanisms by which E1 might potentiate AmB activity were investigated in vitro. When cryptococci were preincubated with AmB or another polyene antibiotic, nystatin, there was an augmented binding of E1. AmB enhanced phagocytosis by unstimulated peritoneal macrophages in the presence of E1 or normal rabbit immunoglobulins. Normal and immune IgG deserve further study to determine under what circumstances the chemotherapeutic effect of AmB can be enhanced.

Improvement of amphotericin B activity during experimental cryptococcosis by incorporation into specific immunoliposomes

Cryptococcosis is an opportunistic infection that is responsible for increased morbidity and mortality in patients with the acquired immunodeficiency syndrome. The high toxicity of the antifungal agent that is mainly used against cryptococcosis, amphotericin B (AMB), accounts for the need for new treatments, especially in patients with the acquired immunodeficiency syndrome because of the high relapse rate of cryptococcosis. Drug targeting may be one of these alternate treatments. Since we have demonstrated that an immunoglobulin G1 (IgG1) anti-Cryptococcus neoformans serotype A monoclonal antibody (E1) was protective during experimental cryptococcosis in mice, we investigated whether specific targeting of AMB with liposomes that bear E1 would improve the therapeutic index of the drug. For that purpose, in vitro and in vivo experiments were designed to compare the specificities and activities of these liposomes with those of control immunoliposomes bearing a nonrelated IgG1 monoclonal antibody (CY34). The immunoliposomes were prepared by covalently linking E1 or CY34 and small unilamellar vesicles. When immunoliposomes were incubated with yeast cells, only E1-bearing liposomes recognized C. neoformans. In vivo, mice that were treated 24 h after infection with one injection of AMB (0.12 mg/kg of body weight) intercalated into E1-bearing liposomes survived significantly longer than did those given the same dose of AMB alone or AMB intercalated into nontargeted liposomes or control immunoliposomes. None of the mice that were given control treatments did statistically better than those that were given AMB. Keeping in mind that this kind of therapy requires knowledge of the antigenic type of the infecting organism, the results suggest that specific targeting of small doses of AMB improve the efficacy of AMB and might be an alternative to the use of larger doses of AMB.

Immunosuppression in experimental cryptococcosis in rats: Modification of macrophage functions by T suppressor cells

The influence of lymphocytes on the modulation of macrophage functions in altered immune states induced by Cryptococcus neoformans infection in rats has been investigated. In this report we observed a decrease of 'in vitro' phagocytic activity by peritoneal cells (PC) from rats that received T suppressor cells induced by cryptococcal infection, against both the same microorganism that stimulated this suppressor population (p less than 0.05) and another non-pathogenic primary yeast (Candida tropicalis), (p less than 0.02). The microbicide function of the PC from these animals present a significant decrease in challenge by C. tropicalis (p less than 0.002) when compared with PC from animals transferred with T normal cells. The transference of T suppressor cells induced by cryptococcal infection in animals immunized with human serum albumin-complete Freund's adjuvant (HSA-CFA) produces a significant alteration of the phagocytosis to HSA-human red cells (HSA-HRC) when compared with the phagocytosis observed in animals that received T normal cells or the phagocytosis of normal animals (p less than 0.001). We could also observe that the DTH to HSA studied during 30 days was negative in rats transferred with PC sensitizated with HSA and treated with suppressor T cells, when compared with the DTH response of animals transferred with PC-HSA cocultured with normal cells (p less than 0.05 21st day). The data presented in this paper illustrated that following infection of rats with C. neoformans there is a change in some population of accessory cells behavior reflected by the modification of several functions, such as phagocytosis, lytic activity and antigen presentation.

Garlic (Allium sativum) in the treatment of experimental cryptococcosis

An extract of garlic was studied for its efficacy in treatment experimental cryptococcosis in mice. The garlic, given by mouth, appeared to reduce brain cryptococcal populations, although the results obtained were generally inconsistent. Complete eradication of the cryptococci was not achieved and the early beneficial results observed diminished over time.

Electron microscopic cytochemical analysis of hepatic granuloma induced by Cryptococcus neoformans.

The hepatic granulomas in experimental cryptococcosis were analyzed by peroxidase (PO) cytochemistry. Cryptococcus neoformans was inoculated intravenously into rats (group A), and some rats were administrated with dextran sulphate to suppress Kupffer cell functions before inoculation (group B). All rats were sacrificed 7 days after inoculation. The livers were examined PO cytochemically. In addition, the liver, spleen, lungs, kidneys and brain were also examined histopathologically. The hepatic granulomas consisted of the following four type cells; exudate macrophages (type I), PO-negative macrophages (type II), Kupffer cells (type III), and other inflammatory cells (type IV) such as neutrophils and lymphocytes. The percentages of the granuloma-composing cells in group A were 10.3% (type I), 27.3% (type II), 52.9% (type III) and 9.5% (type IV), respectively. In contrast in group B, type II cells outnumbered type III cells by a ratio of 5:3. In group B, necrosis and hemorrhage were observed in the granuloma. The lesions in the lungs changed from granulomatous to cystic ones after suppression of the Kupffer cell functions. These results suggest that resident macrophages such as Kupffer cells may play an important role in the formation of cryptococcal lesions.