Elmer Brummer

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. Images

Gamma-interferon activation of macrophages for killing of Paracoccidioides brasiliensis and evidence for nonoxidative mechanisms

Fungicidal activity of murine peritoneal macrophages for the yeast form of the dimorphic fungal pathogen P. brasiliensis was studied. Killing was assessed by reduction of colony forming units (CFU) using a new medium which has a good plating efficiency. Resident peritoneal macrophages phagocytosed but did not kill P. brasiliensis. Macrophages treated overnight with recombinant gamma-interferon (IFN), lymph node cells plus concanavalin A (Con A) or Con A-stimulated spleen cell culture supernatants (Con A Sup) reproducibly killed three different isolates of P. brasiliensis (35 - 55%, P less than 0.05 - P less than 0.001). This is the first demonstration of killing of this organism by macrophages. Activated macrophages did not show enhanced phagocytosis of P. brasiliensis. Activation of macrophages for killing by IFN was dose-dependent and, varying with the isolate, 100 - 10,000 U/ml was required for inducing significant fungicidal effects against P. brasiliensis. Activation of macrophages by IFN or Con A Sup was abrogated by anti-IFN antibody. These results suggest that immune modulation may be an approach to therapy of paracoccidioidomycosis. Killing was not significantly inhibited in the presence of superoxide dismutase (450 U/ml), catalase (20,000 U/ml), dimethylsulfoxide (300 mM) or azide (1 mM). This indicated that killing mechanism(s) did not depend upon products of the oxidative burst. These results show that P. brasiliensis can be significantly killed by activated macrophages without products of the oxidative burst.

Virulence of Paracoccidioides brasiliensis: The influence of in vitro passage and storage

Stability of virulence in P. brasiliensis isolates was studied with respect to the in vitro culture history and methods used for storage. Virulence in yeast-form P. brasiliensis isolates was tested in a chronic pulmonary murine model of paracoccidioidomycosis where progression of disease was quantitated in terms of colony forming units recoverable from lungs. Four isolates of P. brasiliensis, including recently isolated from patients or experimental animals, caused chronic progressive disease. Two isolates with a history of subculturing showed attenuation by causing resolving but chronic disease. An attenuated isolate became avirulent subsequent to 15 more years of subculturing. These findings suggest that virulence of P. brasiliensis can be attenuated or lost subsequent to cycles of subculturing over long periods. Our data suggest that the use of fresh P. brasiliensis isolates may be needed to provide reproducible virulence for experimental systems.

The interaction of human monocytes, monocyte-derived macrophages, and polymorphonuclear neutrophils with caspofungin (MK-0991), an echinocandin, for antifungal activity against Aspergillus fumigatus

The collaboration between human effector cells and caspofungin (MK-0991), a 1,3-beta-D glucan synthase inhibitor, was studied for antifungal activity against Aspergillus fumigatus. Caspofungin was co-cultured for 24h with either human monocytes (Monos), monocyte-derived macrophages (MDM), or polymorphonuclear neutrophils (PMN) against germlings of A. fumigatus and antifungal activity assessed using the XTT metabolic assay. Caspofungin at 0.1 micorg/ml and 0.05 microg/ml or Monos alone against germlings caused significant inhibition. Microscopically this was correlated with less growth and stunted malformed hyphae. The addition of caspofungin at 0.1 microg/ml and 0.05 microg/ml to the monocyte cultures increased antifungal activity. The inhibition of the combination was significantly greater than drug alone (P <.01) and Monos alone (P <.01). MDM against Aspergillus germlings inhibited hyphal growth. The combination of caspofungin at 0.1 microg/ml and 0.05 microg/ml to the macrophage cultures increased antifungal activity. The growth inhibition by the combination was significantly greater than drug alone (P <.01) and MDM alone (P <.01). There was no significant interference with or enhancement of PMNs and caspofungin. These data support the activity of caspofungin against A. fumigatus in vitro, and indicates a cooperative activity with human effector cells. This suggests caspofungin in vivo would have increased efficacy as it combines with host defenses against A. fumigatus.

Activation of Murine Polymorphonuclear Neutrophils for Fungicidal Activity by Recombinant Gamma Interferon

Recombinant murine gamma‐interferon (IFN) was tested for its ability to enhance murine polymorphonuclear neutrophil (PMN) fungicidal activity in vitro. PMNs, elicited by intraperitoneal injection of thioglycollate 4 hr prior to collection, were treated with 0.00003‐300,000 units of IFN per milliliter for 1 hr prior to challenge with yeast form Blastomyces dermatitidis. These PMNs were not fungicidal for Blastomyces in the absence of IFN; significant enhancement of PMN fungicidal activity by IFN treatment occurred in a dose‐dependent manner with maximal enhancement observed at 30,000 U/ml (21% killing). Pretreatment of IFN with rabbit anti‐IFN antiserum before addition to PMNs eliminated the enhancement of fungicidal activity by effective doses of IFN. PMN fungicidal activity against phagocytizable Candida albicans was significantly (P < .001) higher (71.3 ± 17.4%) than against 8. dermatitidis. Candidacidal activity was not significantly enhanced by IFN treatment of PMNs. Exogenously added lipopolysaccharide, at levels corresponding to those found in this preparation of IFN, did not activate PMNs for enhanced fungicidal capacity. These data indicate a stimulatory role for IFN in the killing of 6. dermatitidis by PMNs, suggesting that IFN is an active component of the communication between T lymphocytes and PMNs with respect to antimicrobial resistance. They suggest a natural role for IFN in host defense against blastomycosis and other fungal infections, and a possible therapeutic use for exogenous IFN in fungal disease.

Influence of Human Sera on the In Vitro Activity of the Echinocandin Caspofungin (MK-0991) against Aspergillus fumigatus

ABSTRACT There have been several reports that the activity of echinocandin antifungal agents is not affected or decreased in the presence of human sera. It is known that these drugs are bound >80% in animal and human sera. The activity of the echinocandin caspofungin (MK-0991), a 1,3-β-d-glucan synthase inhibitor, againstAspergillus fumigatus with and without human sera was studied. Conidia of A. fumigatus in microtest plate wells formed germlings after overnight culture in RPMI 1640. Caspofungin was then added with or without serum, and the germlings were incubated at 37°C for 24 h. Human serum (5%) in RPMI 1640 alone did not significantly inhibit the growth of A. fumigatus in vitro. Caspofungin in RPMI 1640 exhibited dose-dependent inhibition, with concentrations of 0.1 and 0.05 μg/ml inhibiting 24.9% +/− 10.4% and 11.7% +/− 3.6%, respectively (n = 10;P < 0.01). The addition of 5% human serum to caspofungin at 0.1 or 0.05 μg/ml increased the inhibition to 78.6% +/− 5.8% or 58.3% +/− 19.2%, respectively (n = 10; P < 0.01 versus controls and versus the drug without serum). Lower concentrations of serum also potentiated drug activity. The effect of human sera was further seen when using caspofungin that had lost activity (e.g., by storage) against A. fumigatus at 0.1 μg/ml. Inactive caspofungin alone demonstrated no significant inhibition of hyphal growth, whereas the addition of 5% human serum to the inactive drug showed 83% +/− 16.5% inhibition (n = 5; P < 0.01). The restoration of activity of caspofungin was seen at concentrations as low as 0.05% human serum. In contrast to prior reports, this study suggests that human serum acts synergistically with caspofungin to enhance its inhibitory activity in vitro against A. fumigatus.

Protection against pulmonary blastomycosis: Adoptive transfer with T lymphocytes, but not serum, from resistant mice

Abstract A murine model of pulmonary blastomycosis, in which the portal of entry is the same as in natural human infection, was used to study resistance to infection subsequent to pulmonary challenge. Lymphoid cells or serum from mice rendered resistant to fatal pulmonary challenge by resolution of a subcutaneous nonlethal infection were tested for ability to adoptively or passively transfer resistance to the pulmonary compartment of naive recipients. As one example, spleen cells (88 × 10 6 ), lymph node cells (17 × 10 6 ), and peritoneal cells (2 × 10 6 ) from resistant mice, but not normal mice, given iv transferred significant resistance to an LD 100 pulmonary challenge of naive recipients. Serum from resistant mice (ELISA titer 1:80) did not transfer protection. T lymphocytes (18 × 10 6 ) were as effective as unfractionated spleen cells (75 × 10 6 ) in the adoptive transfer of resistance. Spleen cells from resistant mice, when treated with anti-mouse T-cell serum plus complement (20 × 10 6 viable cells), failed to transfer resistance. The transfer of resistance with T lymphocytes from resistant to naive mice was T-cell dose dependent.

Regulation by Granulocyte-Macrophage Colony-Stimulating Factor and/or Steroids Given In Vivo of Proinflammatory Cytokine and Chemokine Production by Bronchoalveolar Macrophages in Response to Aspergillus Conidia

Production of the proinflammatory cytokines interleukin (IL)-1 alpha and tumor necrosis factor (TNF)-alpha and of the chemotactic chemokine macrophage inflammatory protein (MIP)-1 alpha by bronchoalveolar macrophages (BAMs) from mice in response to Aspergillus conidia was tested after in vivo administration of saline, dexamethasone, cortisone acetate, granulocyte-macrophage colony-stimulating factor (GM-CSF), or a combination. Dexamethasone suppressed production of IL-1 alpha, TNF-alpha, and MIP-1 alpha; GM-CSF reduced secretion slightly but antagonized dexamethasone suppression when the two were given in combination. Cortisone acetate gave results similar to dexamethasone, but cortisone acetate suppression of BAM responses lasted 7 days, > or = 4 days longer than dexamethasone suppression. The effect of GM-CSF on cortisone acetate suppression lasted at least 7 days. GM-CSF could promote resistance to conidia by maintaining proinflammatory responses.

Collectins and fungal pathogens: roles of surfactant proteins and mannose binding lectin in host resistance

Collectins, collagenous carbohydrate-binding proteins (C-type lectins), are recognized as important factors in non-specific innate immune responses to pathogens. By binding surface carbohydrate structures of pathogens, collectins modify the interaction between pathogens and the immune system. We review the structure of the lung surfactant proteins (SP) SP-A and SP-D, and the serum collectins, mannose binding lectins; the binding of these collectins to pathogen associated molecular patterns or ligands on pathogenic fungi; and the effect of collectin binding to opportunistic and primary fungal pathogens on the interaction with host defense cells, which can result in enhancement or inhibition of resistance. The result of collectin binding to opportunistic fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Pneumocystis) or primary fungal pathogens (Blastomyces dermatitidis, Coccidioides, Histoplasma capsulatum, Paracoccidioides brasiliensis) on interaction with host defense cells relative to complement fixation, phagocytosis, and stimulation of cytokine/chemokine production is reviewed. Increased understanding of these relationships in future will help understand fungal pathogenesis and host defenses against mycoses.

Chronic pulmonary and disseminated paracoccidioidomycosis in mice: quantitation of progression and chronicity

An animal model of chronic paracoccidioidomycosis was established in male adult BALB/cByJIMR mice by intranasal instillation of different doses of yeast form Paracoccidioides brasiliensis. The inoculum was standardized in terms of virulence, age of the culture, percentage of multicellular fungal units containing 1-3 cells, and viability. Progression and chronicity of the infection was measured by quantitative counts of colony forming units (CFU) of P. brasiliensis from infected lungs, spleens, and livers in a newly developed culture medium. The body weight of the mice and the organ weights were also used to monitor the disease process. Infection with several challenge sizes progressed in the lungs until a maximum of 10(7)-10(8) CFU per lung was reached; in general, the higher the inoculum, the sooner this maximum was reached. In mice infected with 2.5 X 10(6) CFU the maximum was reached at 8 weeks, whereas in mice infected with 3 X 10(4) CFU the maximum was reached 14 weeks after infection. Dissemination of the disease progressed until there were 10(6)-10(7) CFU per spleen or liver. The higher the infective dose, the shorter the time required to reach a maximum stable population of yeasts in spleen and liver (12 weeks with inoculum of 2.5 X 10(6) CFU, 18-26 weeks with inoculum of 7.0 X 10(3) CFU). The body weight of mice tended to diminish with time after infection compared to uninfected controls. In contrast, the weight of lung and spleen increased with time after infection. This model of chronic paracoccidioidomycosis permits evaluation of progression of the disease process and of the multiplication of the yeast in organs.