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Biomedical subjects

E Brummer

Publications and source records attributed to E Brummer.

At least 109 records · Page 6Linked to original sources
PubMed1984

Immunological activation of polymorphonuclear neutrophils for fungal killing: studies with murine cells and blastomyces dermatitidis in vitro.

The interaction of elicited murine polymorphonuclear neutrophils (PMN) and the thermally dimorphic fungal pathogen Blastomyces dermatitidis in vitro was studied. The PMN elicited intraperitoneally with thioglycollate, in normal mice or mice immune to B dermatitidis, failed to reduce colony forming units (CFU) of B dermatitidis in the inoculum in a 4-hr in vitro assay, even in the presence of 10% fresh immune serum. In contrast, PMN elicited intraperitoneally in immune mice by injection of nonviable B dermatitidis cells significantly reduced inoculum CFU (60 +/- 5%) under the same conditions. Furthermore, nonviable B dermatitidis intraperitoneally (i.p.) in normal mice or nonviable Candida albicans i.p. in immune mice failed to elicit peritoneal exudate cells that reduced inoculum CFU in this system. These results support the concept that PMN, elicited in a site by means of an immunological reaction, acquired enhanced microbicidal activity. The fungicidal activity of immunologically elicited PMN was shown to be most effective at high effector to target cell ratios (1,000:1), maximal within 2 hr of coculture, and significantly enhanced in the presence of fresh immune serum compared to heat-inactivated immune serum, normal mouse serum, or fetal bovine serum. Such PMN also had significantly enhanced fungicidal activity against C albicans compared to normal PMN. Fungicidal activity was abrogated in the presence of catalase, implicating hydrogen peroxide generation as the killing mechanism in the activated cells.

Animals↗
PubMed1984

The iron-hydrogen peroxide-iodide system is fungicidal: activity against the yeast phase of Blastomyces dermatitidis.

A series of experiments show the potency of a newly described microbicidal system, involving iron, H2O2, and halide, in killing a fungus (Blastomyces dermatitidis). B dermatitidis has previously been shown susceptible to the myeloperoxidase-H2O2-halide system. The present studies show killing of either of two strains in 1 hour if Fe++ at 5 X 10(-5)M, H2O2 at 5 X 10(-5)M and Kl at 5 X 10(-4)M are all present (P less than 0.001). EDTA, a Fe++ chelator, abrogates killing. The mechanism presumably utilizes hydroxyl radical, since an inhibitor, ethanol, also neutralizes the system. The bactericidal and fungicidal system is of great potential importance in vivo.

Antifungal Agents↗
PubMed1984

Toxic effect of products of oxidative metabolism on the yeast form of Paracoccidioides brasiliensis.

The effectiveness of toxic oxygen metabolites in killing the yeast form of Paracoccidioides brasiliensis (the form that occurs in host tissues) was studied with a fluorescence method in vitro. The two isolates studied were similar in susceptibility and H2O2 alone was lethal with an LD50 of 15-25 mM. The addition of halide (5 X 10(-4) M) augmented the lethality of H2O2 and in that setting H2O2 was c.90% lethal at 5 X 10(-5) M. Killing was most effective in the presence of peroxidase, when only 5 X 10(-6) M H2O2 (a concentration attainable in vivo by phagocytes) was required for a 95% kill. Kinetic studies revealed that toxic concentrations of H2O2 alone or of the H2O2-halide-peroxidase (PPH) system produced significant killing in 1 min; killing was maximal in 15 min. The PPH system was the more rapid in action. The dependence of the PPH killing system on H2O2 was demonstrated by an absence of killing in the presence of catalase. The susceptibility of P. brasiliensis to H2O2 and the PPH system appeared different in some respects from that noted for other dimorphic fungal pathogens. These studies suggest that toxic oxygen metabolites are important in host defence against P. brasiliensis.

Catalase↗
PubMed1984

Activation of murine polymorphonuclear neutrophils for fungicidal activity with supernatants from antigen-stimulated immune spleen cell cultures.

An in vitro model of in vivo immunological activation of murine polymorphonuclear neutrophils (PMN) was developed. Culture supernatants of spleen cells from Blastomyces dermatitidis-immunized mice stimulated with B. dermatitidis antigens in vitro were studied. Incubation of the supernatants with thioglycolate-elicited PMN enabled the cells to significantly reduce (31 +/- 6%) B. dermatitidis inoculum CFU. Optimum production of active supernatants occurred after 4 to 6 days of stimulation in vitro and required 200 micrograms of nonviable B. dermatitidis cells per ml. Generation of activity by immune spleen cells was shown to be antigen specific in that stimulation with a heterologous antigen or stimulation of nonimmune spleen cells with B. dermatitidis antigen did not produce active supernatants. The activity in supernatants was dose dependent, nondialyzable (molecular weight greater than or equal to 14,000), and relatively heat labile (80 degrees C, 30 min). Activation of PMN by supernatants for fungicidal activity against B. dermatitidis required only a short incubation period (1 h) followed by a 2-h coculture (challenge) period. Stimulation of normal spleen cells with concanavalin A also resulted in the production of supernatants capable of activating PMN for significant fungicidal activity (31.1 +/- 8.5%). These findings demonstrate for the first time a link between soluble factors produced by antigen stimulation of sensitized lymphoid cells and activation of PMN for enhanced microbicidal activity. Such a process defines an additional immune defense mechanism whereby the immune host may clear specific microorganisms.

Animals↗
PubMed1984

Chronic murine paracoccidioidomycosis: effect of ketoconazole on clearance of Paracoccidioides brasiliensis and immune response.

In a murine model of chronic pulmonary and disseminated paracoccidioidomycosis, ketoconazole (100 mg kg-1 in 0.3% agar) given by gavage twice daily for 1 or 2 months enabled all mice to clear disseminated Paracoccidioides brasiliensis from the spleen. Clearance of P. brasiliensis from the lungs was more difficult, and was achieved in 60% of the mice treated for 2 months. Sera from agar-treated control mice at days 77 and 103 post-infection demonstrated precipitating antibodies to P. brasiliensis antigens, but sera from ketoconazole-treated mice were precipitin-negative, indicating a favorable prognosis. Delayed hypersensitivity reactions to P. brasiliensis antigens in ketoconazole-treated mice were not significantly greater than in controls; consequently this test correlated less well with response than levels of serum antibody. This is the first use of this animal model of paracoccidioidomycosis to study the effect of antifungal drug protocols on the resolution of the disease. It also demonstrates the utility of this model in addressing clinically relevant questions about this disease and its treatment.

Animals↗
PubMed1984

Murine model of paracoccidioidomycosis. Production of fatal acute pulmonary or chronic pulmonary and disseminated disease: immunological and pathological observations.

A model of fatal acute pulmonary or chronic pulmonary and disseminated paracoccidioidomycosis was developed by intranasal challenge of young (3-4-week-old) mice with high doses (2.5-10 X 10(7) units) or low doses (0.1-1 X 10(7)) of yeast-phase Paracoccidioides brasiliensis. Fatal acute paracoccidioidomycosis was dose dependent; 10, 5, and 2.5 X 10(7) viable units of P. brasiliensis produced 100%, 66%, and 17% mortality, respectively, within 11 days. The pathologic picture was that of consolidation with a neutrophil infiltrate. Infection of adult (7-8-week-old) mice with even 10 X 10(7) units did not produce acute fatal paracoccidioidomycosis. The model of fatal acute paracoccidioidomycosis lends itself well to studies of therapeutic intervention. Increasing degrees of chronic pulmonary-disseminated paracoccidioidomycosis were produced by pulmonary infection of young mice with 0.1-1 X 10(7) viable units of P. brasiliensis. Although mice in all groups appeared healthy, pathology in lungs, liver, and spleen was evident at 7 and 10 weeks postinfection. Histopathologic observation revealed acinonodular granulomatous inflammation. At 12 weeks postinfection, there was evidence of less pathology, and of clearing of abscesses. In contrast to the pathology produced by 0.1 X 10(7) P. brasiliensis in young mice, no pathology evident on gross examination was produced by a similar dose in adult mice. Immunological evaluations of mice with chronic pulmonary-disseminated disease showed that spleen cells, but not lymph node cells, had significantly depressed blastogenic responses to concanavalin A (ConA) early after infection. However, at 12 weeks postinfection, when pathological examination indicated beginning resolution of infection, spleen cell responses to ConA were normal. The model of chronic disease is of interest for further immunological studies as well.

Acute Disease↗
PubMed1983

Immunostimulation with muramyl dipeptide and its desmethyl analogue: Studies of non-specific resistance to pulmonary blastomycosis in inbred mouse strains.

N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) protected against pulmonary blastomycosis when given prophylactically to BALB/c mice. Its desmethyl analogue (DM-MDP) had a similar effect. In C3H/HeJ, the effect was less marked. Early treatment after infection, with MDP and DM-MDP, had a modest effect in C3H/HeJ and BALB/c respectively, whereas late treatment had no effect in any mouse strain. No effect could be demonstrated with challenge sizes producing too lethal a model or minimal lethality, or in DBA/2J or young BALB/c mice. The effects in various strains do not correlate with differing effects on nonspecific immunostimulation in these strains. Immunostimulation with glycopeptides deserves further study in prophylaxis or therapy of fungal infection.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1983

Activation of peritoneal macrophages by concanavalin A or Mycobacterium bovis BCG for fungicidal activity against Blastomyces dermatitidis and effect of specific antibody and complement.

With a new short-term assay, where the reduction of CFUs in the inoculum could be measured, we investigated the killing of the dimorphic fungal pathogen Blastomyces dermatitidis in its yeast phase by murine peritoneal macrophages. Peritoneal macrophages from concanavalin A- or Mycobacterium bovis BCG-treated mice, but not resident or thioglycolate-elicited macrophages, significantly reduced the CFUs of B. dermatitidis in the inoculum. The activation of peritoneal macrophages for fungicidal activity by concanavalin A treatment was shown to be dose dependent and transient, i.e., absent after 72 h. These results indicate that it is possible for murine peritoneal macrophages to kill B. dermatitidis in vitro. The addition of specific antibody or complement or both did not enhance the killing of B. dermatitidis by these nonspecifically activated macrophages.

Animals↗
PubMed1983

Susceptibility of Blastomyces dermatitidis strains to products of oxidative metabolism.

Three strains of Blastomyces dermatitidis which differ in their virulence for mice were exposed in their yeast form to various components of the peroxidase-hydrogen peroxide-halide system. Susceptibility to H2O2 alone correlated with virulence, with the most virulent strain (ATCC 26199) least susceptible (50% lethal dose, greater than 50 mM) and an avirulent strain (ATCC 26197) most susceptible (50% lethal dose less than 3.3 mM). A strain of intermediate virulence (ATCC 26198) was of intermediate susceptibility (50% lethal dose, 11.5 mM). The addition of a nontoxic concentration of KI (5 X 10(-4) M) did not increase H2O2 toxicity. However, the addition of either myeloperoxidase or horseradish peroxidase and KI markedly decreased the amount of H2O2 required to kill the organisms, with 100 +/- 0% of all strains killed at 5 X 10(-5) M H2O2 and 97 +/- 4, 100 +/- 0, and 94 +/- 8% of ATCC 26199, ATCC 26198, and ATCC 26197 killed, respectively, at 5 X 10(-6) M H2O2. Kinetic studies with H2O2 alone revealed a delayed onset of killing, but virtually 100% of organisms were killed by 120 min of exposure in all strains. By comparison, the peroxidase-hydrogen peroxide-halide system was 100% lethal for all strains at 1 min. The relatively high concentrations of H2O2 required to kill the yeast phase of B. dermatitidis suggest that H2O2 alone does not account for host resistance to the organism. However, the rapidly lethal effect of the peroxidase-hydrogen peroxide-halide system at physiologically relevant concentrations suggests that this may be one mechanism of host defense to B. dermatitidis.

Animals↗
PubMed1983

Murine pulmonary macrophages: evaluation of lung lavage fluids, miniaturized monolayers, and candidacidal activity.

Optimal yields of pulmonary macrophages (PuM) from lavage of normal murine lungs have been obtained by the study of several different lung lavage fluids. Approximately 1 X 10(6) PuM can be consistently recovered from mice weighing 20 to 30 g, using 15 ml of phosphate buffered saline with 0.1% EDTA, with or without 10% fetal calf serum at 37 degrees C. Use of Terasaki tray wells for monolayer formation has allowed a relatively small number of cells (75,000) to be used to prepare multiple replicate wells from each mouse. Simultaneous functional comparisons with other cell types, e.g., peritoneal macrophages (PeM) were performed using this practical and economical system. Data on the phagocytosis and killing of Candida albicans by PuM and PeM are presented as evidence of the utility of this miniaturized system.

Animals↗
PubMed1983

Enhancing effect of murine polymorphonuclear neutrophils (PMN) on the multiplication of Blastomyces dermatitidis in vitro and in vivo.

The purpose of this study was to determine the effect of murine polymorphonuclear neutrophils (PMN) on the multiplication of the fungal pathogen Blastomyces dermatitidis in vitro and in vivo. With a newly devised method, PMN were obtained in adequate numbers (7 X 10(6) per mouse) and purity (92%) for these studies. In 24 h co-cultures PMN, but not lymph node cells (LNC), enhanced the replication of virulent (V) and avirulent (AV) strains of B. dermatitidis 61% and 34%, respectively. In 72 h co-cultures, multiplication was enhanced even more (V, 180%; AV, 140%), when compared to cultures of B. dermatitidis in medium alone. Viability of PMN was not required, because PMN lysates, but not LNC lysates, were effective in enhancing multiplication. Subcutaneous injection of B. dermatitidis mixed with PMN enhanced multiplication by 90% compared to V alone in a subcutaneous abscess model, whereas mixing with LNC did not enhance multiplication. When AV, a strain which does not by itself replicate in vivo, was injected in vivo with PMN it increased 2.75-fold over 4 days. These findings document the in vivo significance of in vitro enhancement of B. dermatitidis replication by PMN, and support the contention that accumulation and death of PMN in B. dermatitidis lesions may exacerbate this infection in its early stages.

Animals↗
PubMed1982

Opposite effects of human monocytes, macrophages, and polymorphonuclear neutrophils on replication of Blastomyces dermatitidis in vitro.

The purpose of this study was to determine the effects of human monocytes, macrophages, and polymorphonuclear neutrophils (PMN) on the fungal pathogen Blastomyces dermatitidis in vitro. Peripheral blood monocyte monolayers significantly inhibited the replication of a virulent strain (V) and an avirulent strain (AV) of B. dermatitidis by 35 and 28%, respectively. Macrophage monolayers, derived from monocytes by in vitro culturing for 9 days, also inhibited the replication of V and AV in 24-h cocultures; in 72-h cocultures, the inhibition was increased (85 and 88%, respectively). By contrast, PMN stimulated the replication of V and AV in 24-h cocultures (i.e., 45%; AV, 18%) and in 72-h cocultures (V, 68%; AV, 65%). No effect was observed in 2-h cocultures of PMN and B. dermatitidis, even though Candida albicans was killed by PMN in concurrent experiments. PMN stimulated replication of V in a dose-dependent manner, and viability of PMN was not a requirement for the achievement of this effect. These results indicate that monocytes and macrophages significantly inhibited the replication of B. dermatitidis, whereas PMN had an opposite effect. Our findings raise the possibility that these phagocytic cells may have similar opposing effects on the replication of B. dermatitidis in vivo.

Blastomyces↗
PubMed1982

Protection against pulmonary blastomycosis: correlation with cellular and humoral immunity in mice after subcutaneous nonlethal infection.

A model of pulmonary blastomycosis in the mouse, in which the portal of entry is the same as natural human infection, was used to study resistance to challenge after subcutaneous infection. One week after subcutaneous infection, mice were partially resistant to pulmonary challenge, and mice challenged two weeks after infection were resistant. Measurement of cellular and humoral immune responses to Blastomyces dermatitidis antigens after subcutaneous infection showed the following. (i) Delayed-type hypersensitivity appeared 1 week after infection, and responses increased for 3 weeks thereafter. (ii) Proliferative responses in vitro appeared in spleen cells at 1 week and in contralateral lymph node cells at 3 weeks, (iii) Serum antibody, detected by an enzyme-linked immunosorbent assay, appeared 1 week after infection and then increased in titer. (iv) Peritoneal macrophages were activated to inhibit replication of B. dermatitidis in vitro by the first week after infection. Prior subcutaneous infection also resulted in rapid clearing of a second subcutaneous challenge, as well as resistance to a lethal intraperitoneal challenge. This resistance was associated with the development of cell-mediated and humoral immune responses. These data provide a chronological framework for selective transfer experiments.

Animals↗
PubMed1981

Virulence of fungi: correlation of virulence of Blastomyces dermatitidis in vivo with escape from macrophage inhibition of replication in vitro.

Strains of Blastomyces dermatitidis which are virulent (V), attenuated (A), or avirulent (AV) in mice have been identified. Virulence of V has been reported to be associated with its ability to replicate in vivo, causing lethal infections in the lungs or in the peritoneal cavity. We report here that V, but not A or AV, has the ability to replicate in vivo in nonlethal subcutaneous lesions. The possibility that V was able to replicate in vivo by evading primary host defenses was investigated. We studied the interaction of macrophages and strains of B. dermatitidis in vitro. In 24-h cocultures, the replication of all three strains of B. dermatitidis was inhibited by macrophages from: (i) untreated mice (20 to 40%), (ii) mice that had recovered from nonlethal blastomycosis (40 to 60%), (iii) concanavalin A-treated mice (50 to 70%), or (iv) peritoneal exudates elicited by thioglycolate (65 to 80%). However, at 72 h in the first three types of cocultures A and AV remained inhibited, whereas V was not inhibited (0 to 4%). Only the most inhibitory macrophages, those induced by thioglycollate, inhibited V at 72 h, though V was again less inhibited than A or AV at that time. Thus, we show a correlation between virulence of V in vivo and its ability to escape from macrophage inhibition of replication in vitro.

Animals↗
PubMed1981

Strain differences in resistance to infection reversed by route of challenge: studies in blastomycosis.

The inbred mouse strains C3H/HeJ and DBA/1J have been shown to represent the extremes of susceptibility and resistance, respectively, to pulmonary blastomycosis. This pattern was completely reversed when challenge was performed by the intraperitoneal route, whether a virulent or an attenuated strain of Blastomyces dermatitidis was utilized. By a third route (subcutaneous), the differences were insignificant. Inhibition of replication of blastomyces in vitro by macrophages from both strains, before or after activation by subcutaneous infection, was similar.

Animals↗