Lanoconazole, a new imidazole antimycotic compound, protects MAIDS mice against encephalitis caused by Cryptococcus neoformans

Katsunori Furukawa, Hidetaka Sasaki, Richard B. Pollard and Fujio Suzuki*

The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The protective effect of a new antifungal compound, lanoconazole, against Cryptococcus neoformans infection in C57BL/6 mice exposed to LP-BM5 murine leukaemia virus (MuLV) (MAIDS mice) was investigated. Mice were infected intratracheally with C. neoformans, strain 613D, 40 days after infection with LP-BM5 MuLV. They were treated orally with various doses of lanoconazole or with fluconazole 10 mg/kg (a positive control) once daily beginning 1 day after the fungal infection and continuing until the end of the experimental period. The number of C. neoformans cells in the lungs and brains of infected mice was determined. Lanoconazole and fluconazole had a similar inhibitory effect on the growth of C. neoformans in the brains and lungs of normal mice. Whereas lanoconazole inhibited the growth of C. neoformans in the brains and lungs of MAIDS mice, the pathogen grew in the brains of MAIDS mice treated with fluconazole. Lanoconazole reduced the number of C. neoformans in the brains of normal mice treated with a type 2 cytokine mixture, whereas fluconazole did not. A predominance of type 2 T-cell responses was demonstrated in MAIDS mice. Splenic T cells from MAIDS mice, but not those from normal mice, released interleukins 4 and 10 into the culture medium when they were stimulated with an anti-CD3 monoclonal antibody. These results suggest that lanoconazole may have the potential to inhibit the growth of C. neoformans in AIDS patients with a predominance of type 2 T-cell responses.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cryptococcus neoformans is one of the most severe opportunistic pathogens in AIDS patients. Approximately 6–10% of AIDS patients in the USA and up to 30% in Africa are infected with C. neoformans; 90% develop meningoencephalitis.1,2 In immunocompetent individuals, primary lung infections with C. neoformans usually resolve without therapy.3 However, such infections in immunocompromised individuals are more likely to disseminate to the central nervous system and can cause a fatal mycosis. Despite aggressive chemotherapy, the mortality rate in AIDS patients infected with C. neoformans has remained high.4 When AIDS patients with cryptococcal meningoencephalitis were treated with amphotericin B or fluconazole, the meningoencephalitis resolved in one-third, active meningoencephalitis persisted in one-third and nearly one-third died.46

T cells are very important in determining the outcome of fungal infection.79 There are two types of T-cell response, type 1 (type 1 T-helper responses and type 1 CD8+ T-cell responses) and type 2 (type 2 T-helper and type 2 CD8+ T-cell responses).1017 Type 1 T-cell responses are upregulatory cellular immune responses associated with increased concentrations of type 1 cytokines [interleukin 2 (IL-2) and interferon gamma (IFN-{gamma})].1015 However, type 1 T-cell responses are down-regulated by type 2 T-cell responses, which accompany the increased production of type 2 cytokines (IL-4 and IL-10).1015 A shift from type 1 T-cell responses to type 2 T-cell responses is associated with reduced resistance to intracellularly growing microorganisms.10,11,18 A predominance of type 2 T-cell responses in AIDS patients has been observed routinely, and AIDS patients are very susceptible to infection with various intracellularly growing pathogens,19,20 including Candida albicans, Pneumocystis carinii, Mycobacterium avium and herpesviruses (herpes simplex virus types 1 and 2, and cytomegalovirus).1923 We have previously reported the roles of type 2 T-cell responses or type 2 cytokines in determining the severity of C. neoformans-associated encephalitis in MAIDS mice.24 The severity of C. neoformans-associated encephalitis in MAIDS mice could be affected, in part, by inhibiting type 2 T-cell responses. This indicates that type 2 T-cell responses have a pivotal role in determining the severity of encephalitis in MAIDS mice infected with C. neoformans.

Although azole antifungal compounds (e.g. fluconazole and itraconazole) are widely used in treating cryptococcal encephalitis in AIDS patients, mortality and morbidity in these patients remain high.46 Lanoconazole, (±)-(E)- (4-(2-chlorophenyl)-1,3-dithiolan-2-ylidene)-1-imidazolylacetonitrile, is a new imidazole antifungal compound with a broad spectrum of antifungal activity in vitro and in vivo.25,26 We showed, in preliminary studies, that type 2 T-cell responses were decreased in MAIDS mice treated with lanoconazole. After stimulation with an anti-CD3 monoclonal antibody, splenic T cells from MAIDS mice treated with lanoconazole released limited amounts of type 2 cytokines into their culture medium, while large amounts of these cytokines were produced by splenic T cells from MAIDS mice treated with saline.

The present study compared the effects of lanoconazole and fluconazole on the growth of C. neoformans in MAIDS mice.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MAIDS mice

Four-week-old C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME, USA) were infected intraperitoneally with LP-BM5 murine leukaemia virus (LP-BM5 MuLV) at 4.5 x 102 pfu/mouse.27,28 Mice exposed to this amount of LP-BM5 MuLV died within 150 days of infection.29 In our experiments, these mice were used as MAIDS mice 40 days after infection. We have shown previously that splenic lymphocytes from MAIDS mice produce less IL-2 and IFN-{gamma} and more IL-4 and IL-10.24 Type 2 T-cell responses were predominant in these mice.24 All procedures for animal experiments were approved by the Animal Care and Use Committee of the University of Texas Medical Branch at Galveston.

C. neoformans

C. neoformans strain 613 D (ATCC 36556) was obtained from the American Type Culture Collection (Rockville, MD, USA). This strain, an encapsulated serotype D strain with an in vitro generation time of 3.1 h, was originally isolated from human lung and spleen tissue.30 The organisms were grown at room temperature for 48 h on Sabouraud dextrose agar (SDA) plates (5% peptone, 2.5% yeast extract, 1.5% agar), washed with saline, counted by means of a haemocytometer and diluted to the appropriate concentrations. After being given 1 x 108 cells of C. neoformans intratracheally, 10–20% of normal mice and 70% of MAIDS mice died, while a dose of 1 x 106 cells/mouse killed no normal mice and 10–20% of MAIDS mice. All mice in both groups survived at least 3 weeks from the onset of the infection; the first deaths in both groups began to occur 4 weeks after infection.

Reagents

Lanoconazole (Tsumura Co., Tokyo, Japan) was suspended in saline at the appropriate concentrations. Various doses of lanoconazole (3, 10 or 30 mg/kg) were administered orally to mice once daily beginning 1 day after infection until the end of evaluation period. Fluconazole (Pfizer Inc., New York, NY, USA) was dissolved in saline and administered orally to mice at a dose of 10 mg/kg for 20 days beginning 1 day after infection. The fluconazole dose was determined from previous publications.31,32 As a control, infected mice were treated with saline (0.1 mL/mouse). A mixture of murine recombinant IL-4 (Genzyme, Cambridge, MA, USA) and recombinant IL-10 (Genzyme) were administered intraperitoneally 1, 3, 5, 7, 9 and 11 days after the infection. The mixture used in this study contained 5.6, 16.8 and 50.0 ng/mouse of each cytokine. The cytokine doses were determined from a previous publication.24 Monoclonal antibodies for IL-4 and IL-10 were purchased from PharMingen (San Diego, CA, USA).

Intratracheal infection with C. neoformans

Normal and MAIDS mice were anaesthetized with pentobarbital (40 mg/kg ip). A small incision was made through the skin over the trachea. The trachea was exposed and a 28-gauge needle (Becton Dickinson, Franklin Lakes, NJ, USA), bent slightly so that it would follow the trachea more closely, attached to an insulin syringe (Becton Dickinson) was inserted into, and parallel with the trachea. A 50 µL suspension of C. neoformans cells containing 1 x 106 or 1 x 108 cells was injected intratracheally into MAIDS mice (five mice each) or normal mice (20 mice each), respectively. Following inoculation, the skin was closed with surgical adhesive. This procedure caused 10–20% mortality in normal mice (1 x 108 cells/mouse) or MAIDS mice (1 x 106 cells/mouse) 4 weeks after C. neoformans infection. All mice in both groups survived at least 3 weeks after infection with C. neoformans. Three weeks after C. neoformans infection, mice were killed and the number of C. neoformans in the brain and lung tissue was determined by a cfu assay. Briefly, organs removed from infected mice were disrupted with a glass homogenizer and a 10% suspension of each tissue was made with saline. Serial 10-fold dilutions of the homogenates were plated on to SDA plates. Seventy-two hours after incubation at room temperature, the number of colonies on the agar plates was counted. The result was expressed as log10 cfu/organ. When no colonies were observed, the log10 cfu was recorded as 0.

Cytokine production by splenic T cells

Mononuclear cells were prepared from the spleens of mice, as described previously.33,34 Briefly, spleens obtained from normal and MAIDS mice 3 weeks after intratracheal infection with C. neoformans (1 x 106 cells/mouse) were teased through steel mesh to prepare a single cell suspension. To obtain mononuclear cells, this cell suspension was subjected to Ficoll-Hypaque gradient centrifugation (400g for 30 min). To obtain splenic T cells, splenic mononuclear cells (5 x 107 cells/mL) were passed through a T-cell enrichment column (R & D Systems, Minneapolis, MN, USA). When spleen cells that had passed through the column were treated with anti-immunoglobulin antiserum and complement, only a 3% reduction in viable cells was demonstrated. In contrast, treatment of these cells with an anti-CD3 monoclonal antibody (PharMingen, San Diego, CA, USA) and complement caused a 97% reduction in the number of viable cells. These results indicated that the purity of these T-cell preparations was about 97%.

For in vitro induction of IL-4 and IL-10, 2 x 106 cells/mL splenic T cells were stimulated with anti-CD3 monoclonal antibody (2.5 mg/L) for 48 h at 37°C, as described previously.35 An ELISA test (PharMingen) was used to assay IL-4 and IL-10 in the harvested culture fluids. Cytokine titres of tested samples were calculated by using reference amounts of standard recombinant cytokines. The assay was performed three times and the results were expressed as the mean of these tests.

Statistical analyses

Statistical analyses were performed using Fisher's protected least significant difference (PLSD) (StatView; Brain Power, Inc., Calabasa, CA, USA) when the overall analysis of variance (ANOVA) was significant. All data are expressed as means ± s.d. Values of P < 0.05 were considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effects of various oral doses of lanoconazole on the growth of C. neoformans in the brains and lungs of normal mice

Normal mice infected intratracheally with C. neoformans (1 x 108 cells/mouse) were treated with lanoconazole 3, 10 or 30 mg/kg. As positive controls, infected mice were treated with fluconazole 10 mg/kg. Three weeks after infection, the number of C. neoformans in brains and lungs of these mice was determined by a cfu assay. As shown in Figure 1aGo, the growth of C. neoformans in the brains of normal mice was inhibited following oral administration of fluconazole 10 mg/kg or lanoconazole 10 or 30 mg/kg. The log10 cfu of C. neoformans in brain tissue was as follows: saline 0.1 mL/mouse, 3.6 ± 0.1; fluconazole 10 mg/kg, 1.7 ± 0.2; lanoconazole 3 mg/kg, 3.3 ± 0.2; lanoconazole 10 mg/ kg, 1.5 ± 0.2; lanoconazole 30 mg/kg, 1.5 ± 0.3. The growth of C. neoformans was shown in the lungs of normal mice treated with fluconazole 10 mg/kg or lanoconazole 10 or 30 mg/kg (Figure 1bGo). The log10 cfu of C. neoformans in lungs was as follows: saline 0.1 mL/mouse, 4.6 ± 0.1; fluconazole 10 mg/kg, 2.3 ± 0.1; lanoconazole 3 mg/kg, 4.2 ± 0.1; lanoconazole 10 mg/kg, 2.3 ± 0.2; lanoconazole 30 mg/kg, 2.2 ± 0.2. All treatments, except lanoconazole 3 mg/kg, significantly inhibited C. neoformans compared with the saline control. In subsequent experiments, lanoconazole and fluconazole were therefore administered orally to mice at a dose of 10 mg/kg.



View larger version (27K):
[in this window]
[in a new window]
 
Figure 1. Effects of lanoconazole (LCZ) on the growth of C. neoformans in the brains and lungs of normal mice. Normal mice infected intratracheally with C. neoformans (1 x 108 cells/mouse) were treated orally with lanoconazole 3–30 mg/kg or fluconazole 10 mg/kg (FCZ). Three weeks after infection, the number of C. neoformans in (a) the brain and (b) lungs of these mice was determined by a cfu assay. Data are expressed as mean ± s.d. *Significantly different from control mice treated with saline (P < 0.05).

 
Effects of lanoconazole and fluconazole on the growth of C. neoformans in MAIDS mice

The susceptibility of normal and MAIDS mice to C. neoformans infection (1 x 106 cells/mouse) was examined. The results are shown in Figure 2Go. C. neoformans grew much more quickly in the brains of MAIDS mice than in normal mice, but growth rates in the lungs were the same in both normal and MAIDS mice. These results suggest that MAIDS mice were much more susceptible to cryptococcal encephalitis than normal mice.



View larger version (29K):
[in this window]
[in a new window]
 
Figure 2. The growth of C. neoformans in the brains and lungs of MAIDS mice. Normal and MAIDS mice were infected intratracheally with C. neoformans (1 x 106 cells/mouse). Three weeks after infection, the number of C. neoformans in (a) brains and (b) lungs of these mice was determined by a cfu assay. Data are expressed as mean ± s.d. **Significantly different from normal mice (P < 0.001).

 
In the next series of experiments, MAIDS mice infected intratracheally with C. neoformans at a dose of 1 x 106 cells/mouse were treated orally with lanoconazole or fluconazole 10 mg/kg. As shown in the Table, GoC. neoformans numbers in brain tissue from MAIDS mice treated with lanoconazole were the same as those in MAIDS mice treated with saline. However, C. neoformans growth in the brains of MAIDS mice treated with lanoconazole was significantly reduced. The growth of C. neoformans in the lungs of MAIDS mice was decreased equally by either fluconazole or lanoconazole. These results suggest that lanoconazole, but not fluconazole, reduces C. neoformans-induced encephalitis in MAIDS mice.


View this table:
[in this window]
[in a new window]
 
Table. Effects of lanoconazole and fluconazole on the growth of C. neoformans in MAIDS micea
 
The growth of C. neoformans in the brains and lungs of normal mice treated with a mixture of type 2 cytokines

Mice infected with C. neoformans (1 x 106 cells/mouse) were treated intraperitoneally with a mixture of IL-4 and IL-10 (5.6, 16.8 and 50.0 ng/mouse). As shown in Figure 3Go, 3 weeks after the infection, the log10 cfu of C. neoformans in brains of mice was as follows: saline, 0; cytokine mixture 5.6 ng/mouse, 1.6 ± 0.1; 16.7 ng/mouse, 1.8 ± 0.2; 50 ng/ mouse, 2.9 ± 0.1. The growth of C. neoformans in the brains of normal mice was greatly increased in the mice given 50 ng/mouse of the mixture of type 2 cytokines. In the lungs, however, the growth of C. neoformans in mice treated with the various doses of the type 2 cytokine mixture was not significantly different from that in control mice treated with saline (Figure 3Go). These results suggest that type 2 T-cell responses or type 2 cytokines may have a role in the development of cryptococcal encephalitis induced in MAIDS mice.



View larger version (30K):
[in this window]
[in a new window]
 
Figure 3. The growth of C. neoformans in normal mice treated with IL-4 and IL-10. Mice infected intratracheally with C. neoformans (1 x 106 cells/mouse) were treated with a mixture of IL-4 and IL-10 (5.6, 16.7 or 50.0 ng/mouse of each). As a control, mice exposed to the pathogens were treated with saline (0.1 mL/mouse). Three weeks after infection, the number of C. neoformans in (a) the brains and (b) lungs of these mice was determined by a cfu assay. Data are expressed as mean ± s.d. * and **, significantly different from control mice (*P < 0.05, **P < 0.001).

 
Effects of lanoconazole and fluconazole on the growth of C. neoformans in brains and lungs of normal mice treated with type 2 cytokines

Normal mice were infected with C. neoformans (1 x 106 cells/mouse) intratracheally and treated with a mixture of IL-4 and IL-10 (50 ng/mouse, each). These mice were treated orally with lanoconazole or fluconazole 10 mg/kg od for 20 days beginning 1 day after infection. As shown in Figure 4Go, in mice treated with both the mixture of type 2 cytokines and lanoconazole, 1.2 ± 0.2 log10 cfu/organ of the pathogen was detected in brain tissue, while 2.7 ± 0.1 log10 cfu/organ of the pathogen was detected in the brains of mice treated with the type 2 cytokine mixture and fluconazole. These results suggest that lanoconazole can inhibit the growth of C. neoformans in the brains of normal mice treated with the mixture of type 2 cytokines (P < 0.05), while fungal growth was not inhibited in normal mice treated with the mixture of type 2 cytokines and fluconazole. The growth of C. neoformans was equally inhibited in lung tissues of mice treated with the mixture of type 2 cytokines and lanoconazole or fluconazole (P < 0.05) (Figure 4Go). These results suggest that type 2 T-cell responses or type 2 cytokines influence the antifungal effect of fluconazole, but not that of lanoconazole.



View larger version (25K):
[in this window]
[in a new window]
 
Figure 4. Effects of lanoconazole (LCZ) and fluconazole (FCZ) on the growth of C. neoformans in mice treated with a mixture of type 2 cytokines. Mice infected intratracheally with C. neoformans (1 x 106 cells/mouse) were treated with IL-4 and IL-10 (50 ng/mouse of each) and with fluconazole or lanoconazole 10 mg/kg. As a control, infected mice were treated with saline (0.1 mL/mouse). Three weeks after infection, the number of C. neoformans in (a) the brains and (b) lungs of these mice was determined by a cfu assay. Data are expressed as mean ± s.d. {dagger}Significantly different from normal mice (P < 0.001); * and **, significantly different from MAIDS mice treated with type 2 cytokine mixture or with saline (*P < 0.05; **P < 0.01).

 
Production of type 2 cytokines by splenic T cells from MAIDS mice treated with lanoconazole or fluconazole

The influence of lanoconazole or fluconazole on the production of type 2 cytokines by splenic T cells from mice previously exposed to the pathogen was investigated. Normal and MAIDS mice, infected intratracheally with C. neoformans (1 x 106 cells/mouse), were treated with lanoconazole or fluconazole 10 mg/kg for 20 days; splenic T cells harvested from these mice 3 weeks after infection were stimulated with anti-CD3 monoclonal antibody. The results obtained are shown in Figure 5Go. Splenic T cells from C. neoformans-infected normal or MAIDS mice treated with lanoconazole released less type 2 cytokines into their culture fluids. However, splenic T cells from MAIDS mice produced type 2 cytokines whether or not they were infected with the pathogen or treated with fluconazole (Figure 5Go). These results suggest that MAIDS-associated type 2 T-cell responses in MAIDS mice are influenced by the administration of lanoconazole whether or not they are infected with C. neoformans.



View larger version (25K):
[in this window]
[in a new window]
 
Figure 5. The production of type 2 cytokines by splenic T cells from various mice treated with lanoconazole (LCZ) or fluconazole (FCZ). Normal and MAIDS mice, infected intratracheally with C. neoformans (1 x 106 cells/mouse), were treated with a 10 mg/kg dose of lanoconazole or fluconazole. Harvested splenic T cells were stimulated with anti-CD3 monoclonal antibody (2.5 mg/L) and their production of type 2 cytokines was assayed. Data are expressed as mean ± s.d. *P < 0.05 (between groups comparison).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In immunocompetent hosts, cryptococcal infection occurs as an asymptomatic pneumonia and rarely disseminates.36 However, in immunocompromised hosts, the pathogen can disseminate to the central nervous system; the most common life-threatening manifestation of cryptococcosis is meningoencephalitis.37 Therefore, the strategy for treatment of cryptococcal infection is to inhibit the growth of the pathogen in the brains of immunocompromised hosts. Most studies of cryptococcal infection have been in immunocompetent hosts, but the use of immunocompromised hosts will broaden our understanding of the effects of antifungal agents against C. neoformans infection. Approximately 6–10% of patients with AIDS have been infected with C. neoformans;38,39 of these, 90% develop meningoencephalitis. We therefore studied the effect of lanoconazole on the development of meningoencephalitis in MAIDS mice infected with C. neoformans. Lanoconazole, a new imidazole antimycotic compound, shows a broad spectrum of activity against fungi in vitro and in vivo.25,26 In Japan, it has been used clinically in various dermatomycoses, including tinea pedis, and in cutaneous candidosis.

In our study, cryptococcal encephalitis was demonstrated in MAIDS mice within 3 weeks of C. neoformans infection. A 10–20% mortality rate was demonstrated in normal mice exposed to 1 x 108 cells/mouse of C. neoformans, while 70% of MAIDS mice infected with the same amount of the pathogen died. When MAIDS mice were exposed to 1 x 106 cells/mouse of C. neoformans, 10–20% of them died. MAIDS mice were exposed to 1 x 106 cells per mouse of C. neoformans intratracheally and treated with various doses of lanoconazole.

Lanoconazole inhibited the growth of C. neoformans in the brains of MAIDS mice 3 weeks after cryptococcal infection. Fluconazole, which was used as the comparator antifungal agent, did not inhibit the growth of this pathogen in the brains of MAIDS mice. However, both compounds inhibited the growth of C. neoformans equally in the brains and lungs of normal mice 3 weeks after cryptococcal infection. Lanoconazole and fluconazole, which are azole antifungal agents, act by altering cell membranes, resulting in increased membrane permeability, leakage of essential elements and impaired uptake of precursor molecules.40 However, these mechanisms alone may not explain the inhibitory effect of lanoconazole on the growth of C. neoformans in the brains of MAIDS mice. Cell-mediated immunity has been recognized as a key component in determining the outcome of an infection with intracellularly growing organisms.79 In certain intracellular infections, the dissemination of these pathogens has been shown to be associated with the ability of type 2 T cells to produce IL-4 and IL-10 (type 2 cytokines).18,4143 Recently, we have reported on the protective effects of the combination treatment with IL-12 and soluble IL-4R against an established infection of herpes simplex virus type 1 (HSV-1) in thermally injured mice.44 Soluble IL-4R was used as an inhibitor of type 2 T-cell responses and IL-12 was used as an inducer of type 1 T-cell responses. In thermally injured mice treated with soluble IL-4R, IFN-{gamma} production was stimulated by IL-12.44 In this study, C. neoformans grew much more quickly in the brains of MAIDS mice than in the brains of normal mice. A predominance of type 2 T-cell responses in MAIDS mice has been observed previously.28,29 Also, the growth of C. neoformans in brains of normal mice treated with the mixture of type 2 cytokines (IL-4 and IL-10) was markedly greater than that in the brains of normal mice treated with saline. These results suggest that type 2 T-cell responses or type 2 cytokines may play a very important role in the aggravation of cryptococcal encephalitis in mice.

In this study, splenic T cells from C. neoformans-infected MAIDS mice, or those treated with fluconazole, produced type 2 cytokines when they were stimulated in vitro with anti-CD3 monoclonal antibody. However, type 2 cytokines were not produced by splenic T cells from C. neoformans-infected MAIDS mice treated with lanoconazole. These facts suggest that the inhibitory effect of lanoconazole on the MAIDS-associated type 2 T-cell responses may make an important contribution to the anti-cryptococcal activities of the compound in the brains of MAIDS mice.

The effects of antimycotic compounds on the balance of type 1 and type 2 T-cell responses have rarely been investigated. This immunological balance seems to be particularly important in the regulation of certain opportunistic infections. Such infections occur in immunosuppressed individuals whose T-cell responses are dominated by type 2 T-cell responses. A shift from type 1 T-cell responses to type 2 T-cell responses in individuals has also been associated with reduced resistance to certain pathogens, such as cytomegalovirus, influenza virus and HIV.1012 Lanoconazole could thus be useful for treating the meningoencephalitis caused by C. neoformans in AIDS patients.


    Notes
 
* Corresponding author. Tel: +1-409-747-1856; Fax: +1-409-747-1857; E-mail: fsuzuki{at}utmb.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Holmberg, K. & Meyer, R. D. (1986). Fungal infections in patients with AIDS and AIDS-related complex. Scandinavian Journal of Infectious Diseases 18, 179–92.[ISI][Medline]

2 . Dismukes, W. E. (1988). Cryptococcal meningitis in patients with AIDS. Journal of Infectious Diseases 157, 624–8.[ISI][Medline]

3 . Balmes, J. R. & Hawkins, J. G. (1987). Pulmonary cryptococcosis. Seminars in Respiratory Medicine 9, 180–6.[ISI]

4 . Saag, M. S., Powderly, W. G., Cloud, G. A., Robinson, P., Grieco, M. H., Sharkey P. K. et al. (1992). Comparison of amphotericin B with fluconazole in the treatment of acute AIDS-associated cryptococcal meningitis. The NIAID Mycoses Study Group and the AIDS Clinical Trials Group. New England Journal of Medicine 326, 83–9.[Abstract]

5 . Bennett, J. E., Dismukes, W. E., Duma, R. J., Medoff, G., Sande, M. A., Gallis, H. et al. (1979). A comparison of amphotericin B alone and combined with flucytosine in the treatment of cryptococcal meningitis. New England Journal of Medicine 301, 126–31.[Abstract]

6 . Berry, A. J., Rinaldi, M. G. & Graybill J. R. (1992). Use of high-dose fluconazole as salvage therapy for cryptococcal meningitis in patients with AIDS. Antimicrobial Agents and Chemotherapy 36, 690–2.[Abstract]

7 . Kobayashi, M., Kobayashi, H., Mori, K., Pollard, R. & Suzuki, F. (1998). The regulation of burn-associated infections with herpes simplex virus type 1 or Candida albicans by a non-toxic aconitine-hydrolysate, benzoylmesaconine. Part 2: Mechanism of the antiviral action. Immunology and Cell Biology 76, 209–16.[ISI][Medline]

8 . Murphy, J. W. (1989). Cryptococcosis. In The Immunology of Fungal Disease (Cox, R. A., ed.), pp. 93–138. CRC Press, Boca Raton, FL.

9 . Fukazawa, Y., Cassone, A., Bistoni, F., Howard, D. H., Kagaya, K., Murphy, J. W. et al. (1994). Mechanisms of cell-mediated immunity in fungal infection. Journal of Medical and Veterinary Mycology 32, Suppl. 1, 123–31.

10 . Scott, P. & Kaufmann, S. H. (1991). The role of T-cell subsets and cytokines in the regulation of infection. Immunology Today 12, 346–8.[ISI][Medline]

11 . Sher, A., Gazzinelli, R. T., Oswald, I. P., Clerici, M., Kullberg, M., Pearce, E. J. et al. (1992). Role of T-cell derived cytokines in the downregulation of immune responses in parasitic and retroviral infection. Immunological Reviews 127, 183–204.[ISI][Medline]

12 . Del Prete, G. & Romagnani, S. (1994). The role of TH1 and TH2 subsets in human infectious diseases. Trends in Microbiology 2, 4–6.[Medline]

13 . Mosmann, T. R. (1994). Cytokine patterns during the progression to AIDS. Science 265, 193–4.[ISI][Medline]

14 . Mosmann, T. R. & Coffman, R. L. (1989). TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annual Review of Immunology 7, 145–73.[ISI][Medline]

15 . Fitch, F. W., McKisic, M. D., Lancki, D. W. & Gajewski, T. F. (1993). Differential regulation of murine T lymphocyte subsets. Annual Review of Immunology 11, 29–48.[ISI][Medline]

16 . Croft, M., Carter, L., Swain, S. L. & Dutton, R. W. (1994). Generation of polarized antigen-specific CD8 effector populations: reciprocal action of interleukin (IL)-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles. Journal of Experimental Medicine 180, 1715–28.[Abstract]

17 . Cronin, D. C., Stack, R. & Fitch, F. W. (1995). IL-4-producing CD8+ T cell clones can provide B cell help. Journal of Immunology 154, 3118–27.[Abstract/Free Full Text]

18 . Ikemoto, K., Pollard, R. B., Fukumoto, T., Morimatsu, M. & Suzuki, F. (1995). Small amounts of exogenous IL-4 increase the severity of encephalitis induced in mice by the intranasal infection of herpes simplex virus type 1. Journal of Immunology 155, 1326–33.[Abstract]

19 . Horowitz, H. W., Telzak, E. E., Sepkowitz, K. A. & Wormser, G. P. (1998). Human immunodeficiency virus infection, Part II. Disease-a-Month 44, 677–716.

20 . Jacobson, M. A. & French, M. (1998). Altered natural history of AIDS-related opportunistic infections in the era of potent combination antiretroviral therapy. AIDS 12 Suppl. A, S157–63.[Medline]

21 . Odds, F. C. (1988). Candida and Candidosis, 2nd edn, pp. 252–78. Baillière Tindall, London.

22 . Romani, L., Mocci, S., Cenci, E., Rossi, R., Puccetti, P. & Bistoni, F. (1991) Candida albicans-specific Ly-2+ lymphocytes with cytolytic activity. European Journal of Immunology 21, 1567–70.[ISI][Medline]

23 . Maródi, L., Káposzta, R., Campbell, D. E., Polin, R. A., Csongor, J. & Johnston, R. B. (1994). Candidacidal mechanisms in the human neonate. Impaired IFN-gamma activation of macrophages in newborn infants. Journal of Immunology 153, 5643–9.[Abstract/Free Full Text]

24 . Furukawa, K., Sasaki, H., Pollard, R. B. & Suzuki, F. (1998). Pathogenic roles of type 2 T cell responses on Cryptococcus neoformans infection in MAIDS mice. In Clinical Science 2, pp. 129–33. Monduzzi Editore, Bologna.

25 . Niwano, Y., Kuzuhara, N., Kodama, H., Yoshida, M., Miyazaki, T. & Yamaguchi, H. (1998). In vitro and in vivo antidermatophyte activities of NND-502, a novel optically active imidazole antimycotic agent. Antimicrobial Agents and Chemotherapy 42, 967–70.[Abstract/Free Full Text]

26 . Niwano, Y., Tabuchi, T., Kanai, K., Hamaguchi, H., Uchida, K. & Yamaguchi, H. (1995). Short-term topical therapy of experimental tinea pedis in guinea pigs with lanoconazole, a new imidazole antimycotic agent. Antimicrobial Agents and Chemotherapy 39, 2353–5.[Abstract]

27 . Morse, H. C., Chattopadhyay, S. K., Makino, M., Fredrickson, T. N., Hugin, A. W. & Hartley, J. W. (1992). Retrovirus-induced immunodeficiency in the mouse: MAIDS as a model for AIDS. AIDS 6, 607–21.[ISI][Medline]

28 . Kanagawa, O., Vaupel, B. A., Gayama, S., Koehler, G. & Kopf, M. (1993). Resistance of mice deficient in IL-4 to retrovirus-induced immunodeficiency syndrome (MAIDS). Science 262, 240–2.[ISI][Medline]

29 . Sasaki, H., Pollard, R. B. & Suzuki, F. (1997). Effect of thermal injury on the severity of LP-BM5 MuLV infection in mice. In Programme and Abstracts of the Thirty-Seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 1997. Abstract G85, p. 208. American Society for Microbiology, Washington, DC.

30 . Kozel, T. R. & Cazin, J. (1971). Nonencapsulated variant of Cryptococcus neoformans. I. Virulence studies of characterization of soluble polysaccharide. Infection and Immunity 3, 287–94.[ISI]

31 . Nguyen, M. H., Najvar, L. K., Yu, C. Y. & Graybill J. R. (1997). Combination therapy with fluconazole and flucytosine in the murine model of cryptococcal meningitis. Antimicrobial Agents and Chemotherapy 41, 1120–3.[Abstract]

32 . Van Cutsem, J. (1993). Therapy of experimental meningeal and disseminated cryptococcosis. Mycoses 36, 357–67.[ISI][Medline]

33 . Suzuki, F. & Pollard, R. B. (1982). Mechanism for the suppression of gamma-interferon responsiveness in mice after thermal injury. Journal of Immunology 129, 1811–5.[Abstract/Free Full Text]

34 . Kobayashi, M., Herndon, D. N., Pollard, R. B. & Suzuki, F. (1995). CD4+ contrasuppressor T cells improve the resistance of thermally injured mice infected with HSV. Journal of Leukocyte Biology 58, 159–67.[Abstract]

35 . Adkins, B. & Hamilton, K. (1992). Freshly isolated, murine neonatal T cells produce IL-4 in response to anti-CD3 stimulation. Journal of Immunology 149, 3448–55.[Abstract/Free Full Text]

36 . Diamond, R. D. (1984). Cryptococcus neoformans. In Principles and Practices of Infectious Diseases, 2nd edn, (Mandell, G. L., Douglas, R. G. Jr. & Bennett, J. E., Eds), pp. 1460–8. John Wiley & Sons, New York, NY.

37 . Kovacs, J. A., Kovacs, A. A., Polis, M., Wright, W. C., Gill, V. J., Tuazon, C. U. et al. (1985). Cryptococcosis in the acquired immunodeficiency syndrome. Annals of Internal Medicine 103, 533–8.[ISI][Medline]

38 . Holmberg, K. & Meyer, R. D. (1986). Fungal infections in patients with AIDS and AIDS-related complex. Scandinavian Journal of Infectious Diseases 18, 179–92.[ISI][Medline]

39 . Dismukes, W. E. (1988). Cryptococcal meningitis in patients with AIDS. Journal of Infectious Diseases 157, 624–8.[ISI][Medline]

40 . Saag, M. S. & Dismukes, W. E. (1988). Azole antifungal agents: emphasis on new triazoles. Antimicrobial Agents and Chemotherapy 32, 1–8.[ISI][Medline]

41 . Sadick, M. D., Heinzel, F. P., Holaday, B. J., Pu, R. T., Dawkins, R. S. & Locksley, R. M. (1990). Cure of murine leishmaniasis with anti-interleukin 4 monoclonal antibody. Evidence for a T cell-dependent, interferon-gamma-independent mechanism. Journal of Experimental Medicine 171, 115–27.[Abstract]

42 . Leiby, D. A., Fortier A. H., Crawford, R. M., Schreiber, R. D. & Nacy, C. A. (1992). In vivo modulation of the murine immune response to Francisella tularensis LVS by administration of anticytokine antibodies. Infection and Immunity 60, 84–9.[Abstract]

43 . Romani, L., Mencacci, A., Grohmann, U., Mocci, S., Mosci, P., Puccetti, P. et al. (1992). Neutralizing antibody to interleukin 4 induces systemic protection and T helper type 1-associated immunity in murine candidiasis. Journal of Experimental Medicine 176, 19–25.[Abstract]

44 . Kobayashi, H., Kobayashi, M., Utsunomiya, T., Herndon, D. N., Pollard, R. B. & Suzuki, F. (1999). Therapeutic protective effects of IL-12 combined with soluble IL-4 receptor against established infections of herpes simplex virus type 1 in thermally injured mice. Journal of Immunology 162, 7148–54.[Abstract/Free Full Text]

Received 5 August 1999; returned 7 December 1999; revised 9 February 2000; accepted 9 May 2000