Regulation of infection with Histoplasma capsulatum by TNFR1 and -2.

The concerted action of several cytokines is necessary for resolution of both primary and secondary infection with Histoplasma capsulatum. Among the soluble factors that contribute to tissue sterilization, TNF-alpha stands as a central mediator of protective immunity to this fungus. In this study, we explored the regulation of protective immunity by TNFR1 and -2. In primary pulmonary infection, both TNFR1-/- and -2-/- mice manifested a high mortality after infection with H. capsulatum, although TNFR1-/- mice were more susceptible than TNFR2 -/- mice. Overwhelming infection in the former was associated with a pronounced decrement in the number of inflammatory cells in the lungs and elevated IFN-gamma and TNF-alpha levels in the lungs. In contrast, IFN-gamma levels were markedly decreased in TNFR2-/- mice, and treatment with this cytokine restored protective immunity. Lung macrophages from both groups of knockout mice released substantial amounts of NO. Upon secondary infection, TNFR2-/- mice survived rechallenge and cleared infection as efficiently as C57BL/6 animals. In contrast, mice given mAb to TNFR1 succumbed to reexposure, and the high mortality was accompanied by a significant increase in fungal burden in the lungs. Both IL-4 and IL-10 were elevated in the lungs of these mice. The results demonstrate the pivotal influence of TNFR1 and -2 in controlling primary infection and highlight the differences between these receptors for regulation reexposure histoplasmosis.

Vbeta4(+) T cells promote clearance of infection in murine pulmonary histoplasmosis.

T cells are essential for controlling infection with Histoplasma capsulatum. Because the T cell receptor is vital for transducing the biological activities of these cells, we sought to determine if exposure to this fungus induced an alteration in the Vbeta repertoire in lungs of C57BL/6 mice infected intranasally. Vbeta2(+) cells were elevated on day 3 after infection; Vbeta4(+) cells were higher than controls on days 7, 10, and 14 after infection. Vbeta10(+) cells were increased on days 14 and 21, and Vbeta11(+) exceeded controls only on day 14. We investigated the clonality and function of Vbeta4(+) cells because their expansion transpired during the critical time of infection, that is, when cellular immunity is activated. Sequence analysis demonstrated preferential use of a restricted set of sequences in the complementarity-determining region 3. Elimination of Vbeta4(+) cells from mice impaired their ability to resolve infection. In contrast, depletion of Vbeta7(+) cells, the abundance of which was similar to that of Vbeta4(+), did not alter elimination of the fungus. The identification of clonotypes of Vbeta4(+) cells suggests that a few antigenic determinants may drive proliferation of this subset, which is necessary for optimal clearance.

Blockade of endogenous TNF-alpha exacerbates primary and secondary pulmonary histoplasmosis by differential mechanisms.

We investigated the mechanisms by which endogenous TNF-alpha modulates host defenses during experimental primary and secondary pulmonary infection with Histoplasma capsulatum (Hc). Neutralization of TNF-alpha in vivo resulted in increased CFU and 100% mortality in naive and immune mice challenged with Hc intranasally. Levels of IFN-gamma and granulocyte macrophage-CSF were elevated in TNF-alpha-neutralized naive mice, whereas IL-4, -6, -10 and TGF-beta did not differ from controls. In contrast, in secondary histoplasmosis, significant elevations of IL-4 and -10 were observed in TNF-alpha-depleted mice. Alveolar macrophages (Mphi) did not exert fungistatic activity against Hc after exposure to recombinant murine TNF-alpha, recombinant murine IFN-gamma, or both. The increase in susceptibility to primary Hc infection was associated with diminished production of reactive nitrogen intermediates by alveolar Mphi from TNF-alpha-depleted mice, whereas production of nitric oxide during secondary histoplasmosis was similar in both groups. Upon secondary challenge, TNF-alpha-depleted mice were rescued by concomitant neutralization of IL-4 and IL-10, but not either cytokine alone. Thus, TNF-alpha is critical for controlling primary and secondary infection with Hc, and the mechanisms that lead mice to succumb to primary or secondary infection when endogenous TNF-alpha is blocked are different.

Infection with Histoplasma capsulatum: Host-fungus interface.

Histoplasma capsulatum is a pathogenic fungus that has caused infections in all continents except Antarctica although most disease is found within the Americas. It produces a broad spectrum of illness that can on occasion be fatal. Understanding the interaction between the host and the fungus provides insights into the pathogenic mechanisms as well as the host response to replicating fungi. This knowledge can be used to improve treatment as well as diagnosis. Hence, this review summarizes some of the most recent findings regarding host-fungus interaction.

Intrapulmonary response to Histoplasma capsulatum in gamma interferon knockout mice.

We examined the immunobiological responses to Histoplasma capsulatum in lungs of gamma interferon (IFN-gamma) knockout mice (GKO mice). Naive GKO mice succumbed by day 9 to intranasal challenge with 2.5 x 10(6) yeasts, whereas all wild-type (WT) mice survived for 45 days. Compared to lungs of WT mice, the lungs of acutely infected GKO mice exhibited dramatically elevated numbers of CFU in lungs and significantly higher levels of tumor necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not interleukin-12 (IL-12) or IL-4. To determine if IFN-gamma is necessary in reexposure histoplasmosis, GKO and WT mice were inoculated with 10(4) yeasts intranasally and given amphotericin B for 3 weeks. Six weeks later, mice were rechallenged with 2.5 x 10(6) yeasts. All GKO mice died by day 6, whereas all WT mice survived for 45 days. Lungs of GKO mice contained substantially elevated numbers of CFU and higher TNF-alpha and GM-CSF levels but not IL-12 or IL-4. Thus, IFN-gamma is requisite for control of pulmonary histoplasmosis in naive and reexposed mice.

Modulation of immune responses in murine pulmonary histoplasmosis.

The influence of endogenous interleukin (IL)-12 on the course of pulmonary histoplasmosis was examined in naive and immune mice. All naive animals pretreated with anti-IL-12 monoclonal antibody (MAb) died by day 14. All mice died when anti-IL-12 MAb was initiated as late as postinfection day 3. Unlike those of controls, lungs of naive mice given anti-IL-12 MAb had depressed levels of interferon (IFN)-gamma and increased tumor necrosis factor (TNF)-alpha. The 2 groups had similar IL-4 levels. Administration of anti-IL-4 MAb rescued mice from the inimical effects of anti-IL-12 MAb. Survival of mice given both anti-IL-12 and anti-IL-4 MAb was associated with a blunted TNF-alpha response. In reinfection histoplasmosis, treatment with anti-IL-12 MAb did not alter survival. Fungus burden in lungs, livers, and spleens differed at week 2, but not at week 1, of infection. Thus, endogenous IL-12 is critical for optimal generation of a protective immune response in pulmonary histoplasmosis.

Cellular immune responses to recombinant heat shock protein 70 from Histoplasma capsulatum.

Heat shock protein (hsp) 70 from several microbes is antigenic in mammals. In this study we sequenced and expressed the gene encoding this protein from Histoplasma capsulatum to study its immunological activity. The deduced amino acid sequence of the gene demonstrated 71 and 76% identity to hsp7O from humans and Saccharomyces cerevisiae, respectively. A cDNA was synthesized by reverse transcription-PCR and was expressed in Escherichia coli. Recombinant protein reacted with a mouse monoclonal antibody raised against human hsp7O. Splenocytes from C57BL/6 mice immunized with recombinant hsp7O emulsified in adjuvant, but not yeast cells, reacted in vitro to the antigen. Recombinant hsp7O elicited a cutaneous delayed-type hypersensitivity response in mice immunized with protein or with viable yeast cells. Mice were injected with recombinant hsp7O and challenged intranasally with a sublethal inoculum of yeast cells. Vaccination did not confer protection in this model. Thus, recombinant hsp7O can induce a cell-mediated immune response but does not induce a protective response.

Vaccination with recombinant heat shock protein 60 from Histoplasma capsulatum protects mice against pulmonary histoplasmosis.

HIS-62 is a glycoprotein that has been isolated from the cell wall and cell membrane fraction of the pathogenic fungus Histoplasma capsulatum. It is a target of the cellular immune response to this fungus, and it protects mice against a lethal intravenous inoculum of H. capsulatum yeast cells. In this study, we cloned the gene encoding this antigen to reveal its biological nature and studied the immunological activity of recombinant antigen. The amino acid sequences of the NH2 terminus and internal peptides were obtained by Edman degradation. Degenerate oligonucleotides were used to isolate a gene fragment of HIS-62 by PCR. One 680-bp segment that corresponded to the known peptide sequence was amplified from H. capsulatum DNA. This DNA was used to screen a genomic library, and the full-length gene was isolated and sequenced. The deduced amino acid sequence of the gene demonstrated approximately 70 and approximately 50% identity to heat shock protein 60 (hsp 60) from Saccharomyces cerevisiae and hsp 60 from Escherichia coli, respectively. A cDNA was synthesized by reverse transcription PCR and was expressed in E. coli. Recombinant protein reacted with a monospecific polyclonal rabbit antiserum raised against native HIS-62, with monoclonal HIS-62-reactive T cells, and with splenocytes from mice immunized with viable yeast cells. Moreover, vaccination with the recombinant protein conferred protection in mice against a lethal intranasal inoculation with yeast cells. Thus, HIS-62 is a member of the hsp 60 family, and the recombinant hsp 60 is protective against pulmonary histoplasmosis in mice.

Evaluation of SCH51048 in an experimental model of pulmonary aspergillosis.

The efficacy of a novel triazole, SCH51048, was assessed with a murine model of pulmonary aspergillosis and was compared with those of SCH39304 and itraconazole. A wide range of doses of SCH51048 (5 to 50 mg/kg of body weight) was evaluated. Mortality was significantly delayed in mice treated with doses of 5 mg of SCH51048 per kg or greater in comparison with mortality in controls (P < 0.05). Both SCH51048 and SCH39304 at higher doses (30 and 50 mg/kg) reduced the number of viable Aspergillus fumigatus organisms in lung tissue (P < 0.05). In the present model, itraconazole neither delayed mortality nor significantly reduced the counts in tissue at the doses used. We conclude that SCH51048 is an effective therapy for murine pulmonary aspergillosis.

Correlation of in vitro azole susceptibility with in vivo response in a murine model of cryptococcal meningitis.

Correlations between in vitro susceptibility and in vivo responses to fluconazole were sought in a mouse model of cryptococcal meningitis. Twenty clinical isolates were used. Two distinct populations were noted. Eight high-virulence isolates had an LD50 of < or = 252 cfu of Cryptococcus neoformans. Twelve low-virulence isolates had an LD50 of > 252 cfu. For 7 low-virulence isolates, the LD50 was > 20,000 cfu. C. neoformans also had a broad range of in vitro susceptibilities (MICs of 1.25 to > 80 micrograms/mL) at 24 h. A correlation was found between the MIC and the minimum effective dose of fluconazole in mice. This was observed with both survival and tissue counts as parameters of efficacy. This study documents for the first time the in vivo relevance of in vitro susceptibility to an azole antifungal for C. neoformans.

Induction of tumor necrosis factor-alpha in murine Candida albicans infection.

Candidemia in humans is often associated with an endotoxic shock-like syndrome, comparable to gram-negative sepsis. Tumor necrosis factor-alpha (TNF alpha) has been implicated as a mediator in the endotoxic shock syndrome. The possible role of TNF alpha causing early deaths was explored in a murine model of acute infection with Candida albicans. In vitro data from three mouse strains (BALB/c, C3H/HeJ, and C3H/HeN) and in vivo data from BALB/c mice were obtained. Peritoneal macrophages from all three strains produced TNF alpha in vitro when stimulated with C. albicans. After intravenous infection with 10(8) cfu of C. albicans, mice died within 12 h. TNF concentrations in sera from these mice were significantly greater than in controls. Pretreatment of BALB/c mice with anti-murine TNF alpha did not alter mortality of C. albicans-infected mice, but pretreatment with murine TNF alpha reduced mortality. Therefore, in contrast to what was anticipated, TNF alpha may serve a protective role in murine candidiasis.

Transfer of protective immunity in murine histoplasmosis by a CD4+ T-cell clone.

We have reported that a murine Histoplasma capsulatum-reactive CD4+ T-cell line and clones thereof did not adoptively transfer protection against H. capsulatum infection in normal or cyclophosphamide-treated C57BL/6 mice. One explanation for the results was that the T cells failed to traffic to lymphoid organs in these animals. In this study, we have sought to determine whether one of these clones, 2.3H3, could mediate protection in nude (C57BL/10) or irradiated (5 Gy) heterozygous nude (nu/+) C57BL/6 mice. Mice were inoculated intravenously with 10(7) resting 2.3H3 cells or with an equal number of cells of the ovalbumin-reactive clone 1S6; 2 h later, the mice were challenged intranasally with 5 x 10(6) yeast cells. By day 5 of infection, lungs, livers, and spleens of nude and irradiated nu/+ mice given 2.3H3 contained significantly fewer (P < 0.05) CFU than the same organs from mice inoculated with 1S6. This effect was specific for H. capsulatum, since 2.3H3 did not reduce the number of Coccidioides immitis CFU in lungs, livers, and spleens of irradiated nu/+ mice. By day 10, the amounts of H. capsulatum CFU in lungs, livers, or spleens of nude and irradiated nu/+ mice inoculated with 2.3H3 were smaller than those in 1S6-inoculated mice, but these differences did not reach statistical significance (P > 0.05). The mortality rate of mice inoculated with 2.3H3 and that of mice inoculated with 1S6 were similar. Histopathological examination of tissues from 2.3H3- and 1S6-inoculated mice demonstrated the presence of granulomatous inflammation in organs from both groups. Tissues from 2.3H3-treated mice contained fewer yeasts per high-power field than tissues from 1S6-treated mice. Thus, irradiated or nude mice are permissive for the expression of protective immunity by a CD4+ T-cell clone. Although the protective capacity of T cells in these animals is transient, these animals will be useful for differentiating protective from nonprotective T-cell clones.

Coccidioides immitis presenting as a mycelial pathogen with empyema and hydropneumothorax.

A previously healthy Caucasian male developed hydropneumothorax and a pleural peel filled with pleomorphic, septate hyphae. The only organism grown from cultures of the lung and pleural fluid was Coccidioides immitis, confirmed by exoantigen testing. Spherule-endospore forms were produced, however, following injection of the arthroconidial tissue isolate into BALB/c mice. The patient had a positive immunodiffusion complement-fixation test and developed a positive coccidioidin skin test during therapy. He recovered following thoracotomy and wedge resection of the ruptured coccidioidal cavity, and therapy with amphotericin B followed by fluconazole. The sole presence of the mycelial form of the dimorphic fungus C. immitis in the pleural space may have been due to a low CO2 partial pressure at that site secondary to a bronchopleural fistula. The case shows a distinctive and uncommon presentation of coccidioidomycosis which demonstrates the specificity of both the immunodiffusion complement-fixation assay in diagnosing this disease and the exoantigen test in confirming culture results.

Comparison of amphotericin B lipid complex with amphotericin B and SCH 39304 in the treatment of murine coccidioidal meningitis.

To assess the efficacy of amphotericin B lipid complex (ABLC) in the treatment of coccidioidal meningitis, we compared a wide range of doses (0.35-15 mg kg-1, intravenously (IV)) of ABLC with amphotericin B deoxycholate (AmB) (0.3-7 mg kg-1, intraperitoneally (IP)) and (IV) and a new triazole, SCH 39304 (SCH), in an experimental murine model. Survival data showed high dose ABLC to be of equal efficacy to IV and high dose IP AmB and SCH. Quantitative studies confirmed this outcome. No acute toxicity with ABLC, at the doses employed, was found. We conclude that ABLC is effective in the treatment of murine coccidioidal meningitis.

Comparison of SCH 39304 and its isomers, RR 42427 and SS 42426, for treatment of murine cryptococcal and coccidioidal meningitis.

SCH 39304 (304) and its isomers, SCH 42426 (426) and SCH 42427 (427), are new orally administered antifungal azole derivatives. In this study, we compared the efficacy of 304 with that of 426 and 427 in murine models of cryptococcal and coccidioidal meningitis. On day 18 postinfection with Cryptococcus neoformans, controls showed 80% mortality. The 50% protective doses calculated at this day were 0.56 mg of 304 per kg of body weight, 23.5 mg of 426 per kg, and 0.11 mg of 427 per kg. Controls with coccidioidal meningitis all succumbed, and treated mice at the same time point showed 50% protective doses of 10.8 mg/kg for 304, 200 mg/kg for 426, and 2.1 mg/kg for 427. We conclude that isomer 427 is five times as potent, whereas 426 is 1/50th as potent as 304 in these experimental mycoses.

Combined therapy with fluconazole and flucytosine in murine cryptococcal meningitis.

To assess the possible beneficial effects of combined therapy (fluconazole and flucytosine) in the treatment of cryptococcal meningitis in the immunocompromised host, we compared therapy with fluconazole and flucytosine, individually and combined, in the experimental murine model. BALB/c athymic (nu/nu) mice were infected intracerebrally with 150 to 300 CFU of Cryptococcus neoformans. In mortality studies, treatment was initiated 24 h postinfection and continued for 10 to 14 days with either fluconazole (1 to 15 mg/kg of body weight per day), flucytosine (60 to 120 mg/kg/8 h), both drugs, or 0.3% Noble agar (control). Combined therapy delayed mortality significantly when compared with controls and single-drug regimens. This was observed over a broad range of doses. Quantitative determinations of CFU in brain tissue demonstrated a significantly lower burden of C. neoformans in mice receiving combined therapy. The results indicate that combined therapy with fluconazole and flucytosine is superior to single-drug therapy.