Biomedical subjects

E Brummer

Publications and source records attributed to E Brummer.

At least 91 records · Page 5Linked to original sources

PubMed1988

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.

Animals↗

PubMed1988

Impairment of cellular but not humoral immune responses in chronic pulmonary and disseminated paracoccidioidomycosis in mice.

Humoral and cellular immune responses were measured during the progression of chronic pulmonary and disseminated paracoccidioidomycosis in mice. The chronic disease was established by pulmonary infection of mice with different doses of the yeast form of Paracoccidioides brasiliensis isolate GAP. Levels of antibodies to P. brasiliensis, detected in serum by immunodiffusion and enzyme-linked immunosorbent assay, directly correlated with the size of the infectious challenge. Significant delayed-type hypersensitivity (DTH) responses to antigen were largely restricted to week 1 after pulmonary infection with intranasally administered high doses (5.0 x 10(6) or 1.1 x 10(7) CFU per inoculum). In vitro lymphoproliferative responses of peripheral blood lymphocytes (PBL) to P. brasiliensis antigens were significant only at 2 weeks after infection with intranasally administered 1.1 x 10(7) CFU. Responses of PBL to concanavalin A were depressed (50% of control response) as early as 8 weeks and reached a nadir at 10 to 18 weeks after infection. Infected mice made antibodies to sheep erythrocytes (SRBC) (10(9) intravenously [i.v.]) normally at all times tested after infection. In contrast, infected mice sensitized to SRBC (10(6) i.v.) had significantly depressed DTH responses to SRBC at 9 and 20 weeks postinfection compared with noninfected mice. These results indicated that in this model, normal humoral responses developed to homologous and heterologous antigens. In contrast, the T cellular immune responses were depressed with progression and chronicity of the disease. Thus, this model closely mimics the immunological findings in human paracoccidioidomycosis.

Animals↗

PubMed1988

A culture medium for Paracoccidioides brasiliensis with high plating efficiency, and the effect of siderophores.

The plating efficiency of Paracoccidioides brasiliensis on standard mycological media is poor, impairing its isolation and recovery from various sources, particularly infected tissues. We describe a medium that markedly improves P. brasiliensis plating efficiency. It consists of a synthetic medium (modified McVeigh-Morton) supplemented with 4% (v:v) horse serum and 5% (v:v) culture filtrate from stationary phase P. brasiliensis cultures. A commercially available medium (brain-heart infusion), ordinarily inferior to unsupplemented McVeigh-Morton medium, is at least as efficacious as supplemented McVeigh-Morton medium when supplemented in this manner. We show that plating efficiency varies among P. brasiliensis isolates and can even vary with the isolate's history of passage in culture. In contrast, all isolates studied could produce the growth enhancing factors present in culture filtrate. Some siderophores produced by other fungi can be substituted for the culture filtrate, whereas others can be substituted for both the filtrate and serum. The enhancing effect of filtrate and/or serum could be removed by chelating iron. P. brasiliensis-produced siderophores are likely to be the growth enhancing moiety in culture filtrates.

Culture Media↗

PubMed1987

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 less than .001) higher (71.3 +/- 17.4%) than against B. 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 B. 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.

Animals↗

PubMed1987

Effect of a local immune reaction on peripheral blood polymorphonuclear neutrophil microbicidal function: studies with fungal targets.

Peripheral blood polymorphonuclear neutrophils (PMN) from mice immunized with Blastomyces dermatitidis and then stimulated locally (intraperitoneally, ip) with B. dermatitidis antigen had enhanced killing of B. dermatitidis in vitro (54.4 +/- 19.49 of inoculum) compared to nonimmune mice (32.7 +/- 8.7%; P less than 0.02), nonimmune mice given antigen ip (30.6 +/- 14.0%; P less than 0.05), or immune mice not given antigen ip (15.4 +/- 9.9%; P less than 0.01). Peripheral blood PMN from all four groups had marked killing ability against Candida albicans (91.8-99.3% of inoculum). That the killing of B. dermatitidis was due to PMNs was demonstrated by lack of killing by isolated peripheral blood mononuclear cells from all four groups. A local immune reaction can result in enhancement of PMN fungicidal activity, and this is reflected even in peripheral blood PMN. We hypothesize this is an important component of normal host defenses against fungal infection, and likely other microbial infections. Enhancement of PMN microbicidal function by the soluble mediators presumed to be responsible for the effects observed may be an approach to immunomodulating therapy or prophylaxis of infection.

Animals↗

PubMed1987

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.

Animals↗

PubMed1987

Fungicidal mechanisms of activated macrophages: evidence for nonoxidative mechanisms for killing of Blastomyces dermatitidis.

The mechanism(s) by which lymphokine-activated peritoneal macrophages kill Blastomyces dermatitidis was studied. Resident peritoneal macrophages from BALB/cByJ mice, when treated overnight with lymph node cells plus concanavalin A, supernatants from concanavalin A-stimulated spleen cells, or recombinant gamma interferon, were then able to kill a virulent B. dermatitidis isolate (ATCC 26199) (at levels of 25% +/- 4%, 28% +/- 8%, and 21% +/- 5%, respectively). Killing was not significantly decreased or enhanced in the presence of superoxide dismutase (450 U/ml), catalase (20,000 U/ml), dimethyl sulfoxide (300 mM), or azide (1 mM). Viable B. dermatitidis elicited a brisk oxidative burst and superoxide anion production in activated macrophages as measured by lucigenin-enhanced chemiluminescence, e.g., 10(4) cpm. However, these responses were not significantly different from those of control macrophages. Luminol-enhanced chemiluminescence responses by activated or control macrophages were meager (less than or equal to 10(2) cpm). These results indicate that activated macrophages kill B. dermatitidis by a mechanism(s) independent of products of the oxidative burst.

Animals↗

PubMed1987

Effect of murine polymorphonuclear leukocytes on the yeast form of Paracoccidioides brasiliensis [corrected].

The fungicidal activity of murine polymorphonuclear neutrophils from the peripheral blood or elicited intraperitoneally with thioglycollate or with antigen in Paracoccidioides brasiliensis-sensitized [corrected] or nonsensitized mice was studied. Although peripheral blood, thioglycollate-elicited, and antigen-elicited neutrophils from normal mice or thioglycollate-elicited neutrophils from P. brasiliensis-sensitized [corrected] mice killed Candida albicans (57% to 84%), they failed to significantly reduce inoculum colony forming units of P. brasiliensis [corrected] (0% to 13%). In contrast, antigen-elicited neutrophils from sensitized mice reduced colony forming units of P. brasiliensis [corrected] by 40%, and exhibited significantly enhanced candidacidal activity compared to thioglycollate-elicited neutrophils from normal or sensitized mice but not peripheral blood neutrophils from normal mice. Fresh serum, but not specific antibody, was required for optimal killing of P. brasiliensis [corrected], presumably representing an essential role for complement. Killing of P. brasiliensis [corrected] by antigen-elicited neutrophils from sensitized mice correlated with their ability to produce an enhanced oxidative burst, as measured by luminol-assisted chemiluminescence, when interacting with killed P. brasiliensis [corrected] cells. These results indicate that in P. brasiliensis-sensitized [corrected] hosts an inflammatory reaction to P. brasiliensis [corrected] results in activation of neutrophils for significant killing of the pathogen.

Animals↗

PubMed1987

Activation of pulmonary macrophages for fungicidal activity by gamma-interferon or lymphokines.

The ability of murine recombinant gamma interferon (IFN) or lymphokines to enhance the fungicidal activity of murine pulmonary macrophages (PuM) was studied in in vitro. PuM monolayers were incubated overnight with IFN, lymph node cells (LNC) plus concanavalin A, supernatants from Con A stimulated LNC or spleen cell cultures (Con A Sup), or tissue culture medium (TCM) +/- Con A (5 micrograms/ml) or +/- lipopolysaccharide (LPS, 10 ng to 10 micrograms/ml). After treatment, culture fluids were removed and PuM were challenged for 4 h with the yeast-form Blastomyces dermatitidis or 2 h with Candida albicans. Inoculum colony forming units (CFU) of B. dermatitidis were significantly reduced by PuM treated with 1000 U/ml of IFN (25 +/- 3%), Con A Sup (25 +/- 3%) or LNC plus Con A (37-44%), but not by TCM, ConA or LPS. Candida albicans was killed by PuM treated with Con A Sup (33 +/- 8%) or LNC plus Con A (30-43%), but not by TCM, Con A, or LPS, and the activity of Con A Sup was neutralized by anti-IFN antibody. Candida albicans was not significantly killed by PuM treated with IFN doses ranging from 1 to 10(5) U/ml; nor did addition of LPS to IFN, or prolonged (3 day) treatment with IFN, result in significant killing of C. albicans by PuM. However, IFN (100 U/ml) could activate resident peritoneal macrophages for significant candidacidal activity (63%). These data indicate that PuM can be activated for fungicidal activity, and that PuM differ from resident peritoneal macrophages with regard to induction of candidacidal activity by recombinant gamma-IFN.

Animals↗

PubMed1986

An evaluation of the enzyme-linked immunoabsorbent assay (ELISA) for quantitation of antibodies to Paracoccidioides brasiliensis.

The ELISA procedure was adapted for quantitation of antibodies against Paracoccidioides brasiliensis. Using a yeast cytoplasmic antigen and sera from patients with proven paracoccidioidomycosis, we found that 66% of sera reacted at titers greater than or equal to 1:128. Titers of this magnitude were obtained only for 4-5% of sera from healthy blood donors, tuberculosis patients and patients with other systemic mycoses. The exception was sera from patients with histoplasmosis (36% had titers greater than or equal to 1:128). Follow-up of 10 paracoccidioidomycosis patients during the course of therapy indicated a gradual decrease in antibody titers. Because of the technical advantages of the ELISA procedure in comparison with the standard complement fixation test, the ELISA test has potential utility for the quantitative determination of antibodies in patients with paracoccidioidomycosis.

Antibodies, Fungal↗

PubMed1986

Fungicidal activity of murine broncho-alveolar macrophages against Blastomyces dermatitidis.

The fungicidal activity of murine broncho-alveolar macrophages (BAM) against the yeast form of a virulent strain of Blastomyces dermatitidis was studied in the small-volume wells of a Terasaki plate. In a 4-h fungicidal assay, significant killing (16-33%) of the fungus by unstimulated BAM was demonstrated with BAM from normal mice and from mice rendered immune to lethal infection with B. dermatitidis. No significant differences between the activity of BAM from these two sources could be identified. Addition of 10% autologous normal or immune serum did not augment the macrophage fungicidal activity. Simultaneous experiments with peritoneal macrophages (PM) also gave reproducible killing of the yeast form in the wells of the Terasaki plate, but in the larger wells of microtitration plates, PM showed no significant fungicidal activity. On the other hand, BAM had similar fungicidal activity against B. dermatitidis in Terasaki and microtitration-plate wells. The modest fungicidal activity of BAM from immune mice against B. dermatitidis suggests that the resistance of immune mice to respiratory challenge is likely to be based on some augmentation of this first line of defence.

Animals↗

PubMed1986

Fungicidal activity of murine inflammatory polymorphonuclear neutrophils: comparison with murine peripheral blood PMN.

A characteristic of inflammation is the influx of polymorphonuclear neutrophils (PMN) from peripheral blood to the inflammatory reaction. We report on metabolic alterations and alterations in fungicidal activity in PMN elicited intraperitoneally with different inflammatory agents. The fungicidal activity of murine peripheral blood PMN (PB-PMN) against phagocytosable Candida albicans and nonphagocytosable Blastomyces dermatitidis was compared to that of murine inflammatory PMN. PMN elicited with sodium caseinate exhibited enhanced killing of B. dermatitidis (93 +/- 3%) compared to PB-PMN (38 +/- 11.7%). In contrast, thioglycollate medium elicited PMN had significantly less ability to kill B. dermatitidis (3 +/- 5%) than PB-PMN. Incubation of caseinate PMN with thioglycollate medium for 1 h significantly reduced their ability to kill B. dermatitidis (95% vs 25%). This effect was not due to cytotoxicity of thioglycollate medium for PMN. Candidacidal activity of inflammatory PMN (caseinate or proteose peptone-elicited) was not significantly greater than that of peripheral blood PMN. However, inflammatory PMN had significantly greater candidacidal activity than thioglycollate-elicited PMN.

Animals↗

PubMed1985

Mechanisms in opposite modulation of spleen cell and lymph node cell responses to mitogens following muramyl dipeptide treatment in vivo.

It has been reported that a muramyl dipeptide (MDP) treatment regimen (200 micrograms MDP per mouse, Days -4, -3, -2, and -1) that, given prophylactically, affords protection against several infectious agents also induces lymph node hyperplasia, lymph node cell (LNC) hyperresponsiveness to mitogens, and spleen cell hyporesponsiveness to mitogens. The purpose of the present work was to extend those studies and delineate cellular mechanisms involved in these phenomena. It has been found that hyperresponsiveness of LNC was prolonged (7 days) posttreatment; in contrast, hyporesponsiveness of spleen cells was transient and rebounded by Day 4 posttreatment. Hyperresponsiveness of LNC and hyporesponsiveness of spleen cells actively enhanced and depressed normal lymphoid cell responses, respectively, in cell mixing experiments. Hyporesponsiveness of spleen cells was associated with the plastic-nonadherent, non-B-cell fraction and nylon wool-nonadherent subpopulations. Indomethacin (10(-6) M) did not abrogate hyporesponsiveness of spleen cells. These data suggest that splenic suppressor T cells result from MDP treatment and were responsible for spleen cell hyporesponsiveness. On the other hand, hyperresponsiveness of LNC was associated with the nylon wool-adherent cell subpopulations and a higher percentage of nonspecific esterase-positive cells. Hyporesponsiveness of spleen cells was associated with deficient production of interleukin 2 (IL-2), but not of interleukin 1 (IL-1). In contrast, hyperresponsiveness of LNC was not explained by enhanced IL-1 or IL-2 production.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1985

Activities of the modified polyene N-D-ornithyl amphotericin methyl ester and the azoles ICI 153066, Bay n 7133, and Bay l 9139 compared with those of amphotericin B and ketoconazole in the therapy of experimental blastomycosis.

We studied the efficacy of new experimental antifungal drugs, which represent molecular modifications of present active agents, in a murine model of blastomycosis. Ketoconazole, previously the best azole drug studied and which is protective when administered orally, was superior to a new oral imidazole, Bay l 9139, and a new oral triazole, Bay n 7133. A new oral triazole, ICI 153066, was markedly more effective than ketoconazole and is the only oral drug studied which came close to producing complete sterilization of all visceral infection in all animals treated. Amphotericin B, a polyene given parenterally, was shown to be more efficacious than any drug studied. It completely sterilized the infection. A modified polyene, N-D-ornithyl amphotericin methyl ester, was only slightly less effective on a milligram-per-kilogram basis.

Amphotericin B↗

PubMed1985

Enhanced oxidative burst in immunologically activated but not elicited polymorphonuclear leukocytes correlates with fungicidal activity.

Polymorphonuclear neutrophils (PMN) induced locally in immune mice by intraperitoneal injection of antigen exhibit enhanced fungicidal activity compared with PMN elicited with thioglycolate. The mechanism of the differences in these PMN populations was studied. Sublethal infection was used to produce immunity to Blastomyces dermatitidis. A correlation was sought between the ability of PMN to kill, or not kill, B. dermatitidis and the production of the oxidative burst, as measured by luminol-enhanced chemiluminescence (CL). Although elicited PMN cocultured with Candida albicans produced a burst of CL and were candidacidal, killing did not occur when PMN were cocultured with B. dermatitidis. Lack of killing of B. dermatitidis by elicited PMN correlated with lack of stimulation of a brisk oxidative burst. In contrast to elicited PMN, PMN induced by B. dermatitidis antigen responded to this fungus with a burst of CL and a significant reduction of inoculum CFU (80%). Furthermore, these PMN when cocultured with C. albicans produced an enhanced burst of CL, and killing was enhanced compared with that by elicited PMN, e.g., 86 versus 58%. The CL burst and killing of B. dermatitidis by antigen-induced PMN was abrogated in the presence of catalase, implying a critical role for hydrogen peroxide. Partial but significant depression of CL and killing in the presence of dimethyl sulfoxide, a hydroxyl radical scavenger, identified hydroxyl radical, or its metabolites, as a toxic product(s) responsible for a significant fraction of fungicidal activity. These results indicate that the metabolic activity and microbicidal activity of PMN can be altered (enhanced) at the site of an immunological reaction and thus could constitute an important factor in resistance.

Animals↗

PubMed1985

Recombinant and natural gamma-interferon activation of macrophages in vitro: different dose requirements for induction of killing activity against phagocytizable and nonphagocytizable fungi.

Recombinant murine gamma-interferon (IFN) and supernatants from concanavalin A (ConA)-stimulated spleen cells were tested for their ability to activate resident peritoneal macrophages (M phi) for fungicidal activity. M phi monolayers pulsed overnight with IFN exhibited significantly enhanced fungicidal activity against Candida albicans (44 +/- 12 versus 0.0%) and Blastomyces dermatitidis (34 +/- 1 versus 3 +/- 3%). The effect of IFN was dose dependent; however, less IFN (10 U/ml) was required to activate M phi to kill phagocytizable C. albicans than to kill nonphagocytizable B. dermatitidis (1,000 U/ml). ConA-stimulated spleen cell supernatants were also able to activate M phi for fungicidal activity against both fungi. The capacity of ConA-stimulated spleen cell supernatants to activate M phi for fungicidal activity was neutralized in the presence of antibody to murine IFN. ConA-treated monolayers acquired the ability to kill C. albicans, but not B. dermatitidis, which was shown to be associated with residual (10%) lymphocytes in the monolayers. Lipopolysaccharide (0.001 to 10 micrograms/ml) failed to consistently activate M phi for fungicidal activity. These data show that IFN can exert an immunoregulatory role on M phi defense against these fungal pathogens.

Animals↗

PubMed1985

Study of current and new drugs in a murine model of acute paracoccidioidomycosis.

A new murine model of acute paracoccidioidomycosis, whose features include pulmonary infection with dissemination, was used to study the efficacy of currently available drugs and new agents which might be useful clinically. Two oral imidazole drugs, ketoconazole (KTZ) and Bay l 9139 (l9); two oral triazoles, Bay n 7133 (n7) and ICI 153,066 (ICI); and two polyenes, amphotericin B (AMB) and N-D-ornithyl-amphotericin B methyl ester, were studied. KTZ was superior to n7 and l9, particularly after a less lethal challenge. The polyenes could diminish dissemination, but had only modest effects on the pulmonary infection; AMB appeared slightly more effective. ICI was the most effective drug studied, approximately 5-10 times more potent than KTZ on a milligram/kg body weight basis; it had marked effects on both pulmonary and disseminated disease. These initial applications of the model to therapeutic evaluation suggest sufficient flexibility and utility, with information obtainable after relatively short experiments.

Amphotericin B↗

PubMed1985

Treatment of paracoccidioidomycosis with itraconazole in a murine model.

A new triazole, itraconazole, was studied as oral therapy of paracoccidioidomycosis in a murine model. The Paracoccidioides brasiliensis isolate, susceptible to itraconazole in vitro, was given by intranasal challenge, producing acute pulmonary and disseminated disease. Therapy was given twice daily over 4 weeks, and animals observed over 2 months. The infection was lethal for 70-80% of controls (untreated or polyethylene glycol diluent), whereas all treated animals, given 10-200 mg kg-1 day-1, survived. Itraconazole was ineffective in eradicating lung disease in survivors, though effective in treatment of disseminated sites. Since the highest doses did not give a better response than the lower doses, pharmacokinetic studies were performed. These showed irregular curves and small increases in peak serum concentrations and total area under the serum concentration-time curves, which were not proportional to the dose. This non-linearity appears to be best explained by poor absorption. Itraconazole, from these studies, appears to have promise for the therapy of human paracoccidioidomycosis but possibly with a different formulation.

Animals↗