830
Eur. J. Clin. Microbiol. Infect Dis.
pie IMactam resistance of Enterobacter cloacae. Antimicrobial Agents and Chemotherapy 1985, 27: 455--459. 7. Holmes DS, Quigley M: A rapid boiling method for the preparation of bacterial plasmids. Analytical Biochemistry 198I, 124: 193-197. 8. National Committee for Clinical Laboratory Standards:
9. 10, 1I.
12.
Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. M7-T2. NCCLS, Villanova, PA, 1988. Sanders CC, Sanders WE:Type 1 I]-lactamases of gramnegative bacteria: interactions with IMactam antibiotics. Journal of Infectious Diseases 1986, t54: 792-800. Miller GH, Sabatelli FJ, Hare RS, Waltz JA: Survey of aminoglycoside resistance patterns. Developmental and Industrial Microbiology 1980, 21: 91-104. Ames GF: Resolution o[ bacterial proteins by polyaerylamide gel elcctrophoresis on slabs. Membrane, soluble, and periplasmic fractions. Journal of Biological Chemistry 1972, 249: 634-644. Hopkins JM, Towner KJ: Enhanced resistance to cefotaxime and imipenem associated with outer membrane protein alterations in Enterobacter aerogenes, Journal of Antimierobial Chemotherapy 1990, 25: 49-55.
Degranulation of Human Neutrophils after Exposure to Bacterial Phosphofipase C T.K. W a z n y 1, N. M u m m a w 1, B. Styrt 1'2. Because an endogenous phospholipase C (PLC) participates in neutrophil activation and because many bacterial pathogens produce PLCs, these studies examined the effect of PLC from Bacillus cereuson the release of the granule enzyme lysozyme from human neutrophils.Bacillus cereusPLC caused dose-dependent lysozyme release, and combined stimulation ofneutrophiis with PLC and fluoride led to increased secretion. Stimulation of neutrophil degranulation is a potential contributing factor for tissue damage in infections caused by PLC-producing organisms.
products released by bacteria, some of which resemble participants in the neutrophil's intrinsic activation pathway. For example, endogenous phospholipase C (PLC) is involved in neutrophil activation, and C-type phospholipases with varying substrate specificities are also produced and released as exoenzymes by many bacteria including Bacillus cereus (1). In the experiments reported here, we studied the effect of PLC from Bacillus cereus on human neutrophil degranulation. Materials and Methods. Histopaque 1077, two preparations of phospholipase C from Bacillus cereus (type III product no. P6315 and type XIII product no. P5527), Micrococcus lysodeikticus, lysozyme, sodium pyruvate, and NADH-Na 2 were obtained from Sigma Chemical, USA. Neutrophils were isolated from heparinized human blood as done in other studies (2) by Hypaque-Ficoll density gradient centrifugation, and contaminatingerythrocyteswere lysed with hypotonic NaCI solution. Trypan blue dye exclusion was used for evaluating viability of neutrophils. Three gi trypan blue (0.4 % solution in 0.9 % NaC1) was added to 3 gl of cell suspension and the percentage of cells stained by trypan blue was determined immediately by light microscopy. To measure degranulation, stimuli were added to 2 ml of cells in Hanks balanced salt solution, which were incubated in a shakingwater bath at37 °C for 30 min. The tubes were then vortexed briefly and a drop was removed for assessment of cell viability. One ml was removed from each tube, centrifuged at 200 g and 4 °C for 10 min,and the supernatant saved for enzyme assays. The residual 1 ml was lysed with 20 gl 10 % Triton X-100 for determination of total enzyme content. As a marker of degranulation we measured lysozyme, a component of both azurophil and specific granules, by lysis of Micrococcus lysodeikticus as described (2). Results represent lysozymereleased into the media as a percentage of the total lysozyme released by Triton, subtracting spontaneous release in the absence of stimuli. In selected experiments lactate dehydrogenase (LDH) was determined by measuring conversion of NADH into NAD during reduction of pyruvate into lactate. Results and Discussion. When lysozyme release was
Neutrophils kill microorganisms by ingesting them and releasing oxygen metabolites and lysosomal enzymes into the phagocytic vacuole. However, if lysosomal enzymes are released outside the cell, this can result in host tissue damage. Stimuliwhich might cause neutrophil degranulation in vivo include 1Department of Medicine, and 2Department of Microbiology and Public Health, B220 Life Sciences Building, Michigan State University, East Lansing, Michigan 48824-1317, USA.
measured as a marker of degranulation, exposure to two concentrations of PLC type III caused marked enzyme release with a dose response effect (Figure la). When we tested whether PLC directly influenced the lysozyme assay, adding a comparable amount of PLC to standard concentrations of lysozyme did not produce any significant changes in lysis of Micrococcus lysodeikticus. Therefore, we concluded that our Iysozyme measurements accurately indicated granule content release. To define whether PLC
Vol____~9, 1990
831
4O
¢
zo'
01i to PLC unitstml
0nn 5
10 15 20 mM NaF
Figure I: Percent of total lysozyme released after incubation with (a) 0.1 unil/ml type III PLC (n = 8) or 1 unit/ml type III PLC (n = 4), a n d (b) indicated concentrations of sodium fluoride (n = 2 or3 at each concentration).
DbO.h 20' a
~o
Figure 2" Percent of total lysozyme released in response to (a) 0.1 unit/ml type 111 PLC, 12.5 mM NaF or combined stimuli (n = 8), and (b) 1 unit/ml type XIII PLC, 12.5 mM NaF, or combined stimuli (n = 7).
affects neutrophil responses to other stimuli of degranulation, we used sodium fluoride. Lysozyme release after exposure to NaF alone was dose-dependent (Figure lb). However, the highest concentrations tended to cause damage of a large pereentage of ceils as measured by trypan blue staining. We selected concentrations of 12,5 mM for our experiments, in order to stimulate significant degranulation without killing the cells. Figure 2a demonstrates lysozyme released after exposure to PLC alone, NaF alone, and the combined stimuli. The release produced by combined stimuli was significantly higher (p < 0.05 by t-test) than the calculated sum of releases elicited by NaF alone and PLC alone. Experiments using 1 U/ml type XIII PLC showed additive but no synergistic activity in combination with fluoride (Figure 2b). PLC stimulated degranulation was very low in these experiments, possibly due to the use of the different PLC preparation. To determine whether lysozyme release in our experiments could be explained by cell death
instead of active secretion, we measured cytosol lactate dehydrogenase release and trypan blue dye exclusion. Lactate dehydrogenase proved to be an unreliable indicator of neutrophil death because of variable contribution from lysis of contaminating erythrocytes in the neutrophil preparation. Erythrocyte lysis was obvious as pink supernatants in some samples with substantial erythrocyte contamination, and it was not thought practicable to eliminate erythrocytes entirely from the neutrophil preparations because of the risk of leukocyte damage from more vigorous isolation procedures. Because of these problems we based our measurement of leukocyte viability on trypan blue staining which we have found to be comparable in sensitivity to L D H release in other studies (3). More than 95 % of these cells were viable after stimulation, when measured by this method. Therefore, we concluded that lysozyme release caused by PLCwas most likely due to secretion of granule contents from viable cells. To see whether additional lysozyme release could be achieved by even higher phospholipase concentrations, separate experiments were performed using a final concentration of 10 units per ml of Type III PLC. In these three experiments, PLC stimulated release of 71.6 + 1.7 % of total cellular lysozyme. Cells stimulated by PLC showed marked clumping on microscopic examination, making morphologic assessment difficult. A slightly higher percentage of ceils failed to exclude trypan blue, suggesting some cytotoxicity at the very high concentrations, but there was only 2.3 + 1.4 % excess cell death attributable to PLC. Addition of autologous plasma (10 % volume of approximately 1:1 plasma:PBS mixture from the cell separation) did not protect against either the degranulating effect or the cytotoxicity of PLC. Phospholipase C, an important enzyme in the turnover of phospholipids, appears to be an important endogenous mediator of neutrophil activation (4). Several studies suggest that exogenous PLC may also activate neutrophils. Most of the available studies concentrate on activation of the respiratory burst by bacterial phospholipases (5, 6), although phospholipase C obtained from Clostridium welchiiwas shown to activate both the respiratory burst and modest secretion of specific granule contents (7). Contradicting data showing reversible inhibition of the respiratory burst have also been reported (8). Since the respiratory burst and degranulation are frequently but not invariably linked, and the PLCs of Clostridium perfringens and Bacillus cereus may differ substantially in clinicat importance, we thought that investigation of each of these responses to Bacillus cereus PLC would be informative. Although
832
usually of low virulence, Bacillus cereus has shown potential for tissue invasion, cavitation and eschar formation in some settings (9, 10), suggesting that exotoxins might be of adjunctive importance in special situations. Results of our experiments, using phospholipase C from Bacillus cereus, demonstrated activation of neutrophils to release lysosomal contents and enhancement of the effects of a second stimulus. While it seems unlikely that PLC concentrations comparable to those causing degranulation would be achieved in circulation, we would expect that extremely high concentrations of bacterial exoproducts may be encountered by phagocytes in close contact with large numbers of bacteria at a focus of infection, so that these results have potential relevance to clinical situations. Although contributions from possible contaminants of the preparation cannot be altogether ruled out, neutrophil activation by other contaminating Bacillus cereus products could also be physiologically relevant. We did see degranulation in response to two different Bacillus cereus PLC preparations, suggesting that PLC is likely to be the agent producing the observed results. While in vivo confirmation has yet to be obtained, these findings suggest that release of neutrophil lysosomal enzymes may contribute to pathological changes caused by phospholipase-Cproducing organisms.
References 1. AvigadG: Microbialphospholipases.In: Bernheimer AW (ed): Mechanisms in microbial toxinology.John Wiley, New York, 1976,p. 99-161. 2. Styrt B, Johnson PC, Klempner MS: Differentiallysis of plasma membranes and granules of human neutrophils by digitonin. Tissue and Cell 1985, 17: 793-800. 3. Styrt B, Walker RD, White JC: Neutrophil oxidative metabolism after exposure to bacterial phospholipase C. Journal of Laboratory and Clinical Medicine 1989, 114: 51-57. 4. Verghese MW,Charles L, Jakoi L, Dillon SB, Snyderman R: Role of a guanine nucleotide regulatory protein in the activation of phospholipase C by different chemo-attractants. Journal of Immunology,1987, 138:4374-4380. 5. Patriarca P, Zatti M, Cramer R, Rossi F: Stimulation of the respiration of polymorphonuclear leucocytes by phospholipase C. Life Sciences 1970,9: 841.849. 6. Stevens DL, Mitten J, Henry 12."Effects of alpha and gamma toxins from Clostridium perffmgens on human polymorphonuclear leukocytes. Journal of Infectious Diseases 1987, 156: 324-333. 7. Grzeskowiak M, Bianea VD, DeTogni P, Papini E, Rossi F: Independence with respect to Ca2÷ changes of the neutrophil respiratory and secretory response to exogenous phospholipase C and possible involvement of diacylglyceroland protein kinase C. Biochimica et Biophysica Acta 1985,844: 81-90.
Eur. J. Clin. Microbiol. Infect Dis.
8. Gordon LL, Lee SN, Homan A, Prachand S, Weitzman SA: Phospholipase C reversibly inhibits respiratory burst activity in human neutrophils. Clinical Research 1987, 35: 850A. 9. Davey RT, Tauber WB: Posttraumatic endophthalmitis: the emergingrole of Bacillus cereus infection. Reviewsof Infectious Diseases 1987,9: 110-123. 10. SlimanR, Rehm S, Shlaes DM: Serious infectionscaused by Bacillus species. Medicine 1987,66: 218-223.
Inactivity of Terbinafine in a Rat Model of Pulmonary AspergUlosis H. J. Schmitt 1' 2,, j. A n d r a d e 1, F. E d w a r d s 1, Y. Niki 1, E. B e r n a r d 1, D. A r m s t r o n g 1 In a m o d e l of b r o n c h o p u l m o n a r y aspergillosis lerbinafine did not improve survival o f experimental animals in doses up to 80 m g/kg/day despite adequate lung concentrations. Pretreatment and aerosolization of the c o m p o u n d were also ineffective. Terbinafine was markedly less active in vitro when serum was used instead of Yeast-Nitrogen-Glucose-broth. It is concluded that a lack of bioavailability in the presence o f s e r u m m a y explain the lack of activity ofterbinafine in experimental aspergillosis.
Allylamines are a new class of agents which exhibit antifungal activity by inhibiting squalene epoxidase, a key enzyme in ergosterol biosynthesis of fungi (1-4). In vitro, antifungal activity of these agents has been demonstrated against a broad spectrum of fungi (5-7). We previously found that terbinafine was highly active against clinical isolates of A s pergillus spp. (8). Since the options for treatment of patients with pulmonary aspergiilosis are not very satisfactory in terms of efficacy and toxicity (9, 10), we evaluated terbinafine in a standardized model of bronchopulmonary aspergillosis. Materials and Methods. Terbinafine was a gift from
Sandoz, Switzerland. For treatment of experimental animals, terbinafine was initially dissolved in dimethyl sulfoxide (DMSO) and further dilutions were made in sterile saline just prior to intraperitoneal (i.p.) administration. D M S O (5 %) and 0.02 % iInfectious Diseases Service, Department of Medicine and Microbiology Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. 2Children's Hospital, Johannes Gutenberg University, Langenbeckstral3e 1, 6500 Mainz, FRG.
Eur. J. Clin. Microbiol. Infect Dis.
pie IMactam resistance of Enterobacter cloacae. Antimicrobial Agents and Chemotherapy 1985, 27: 455--459. 7. Holmes DS, Quigley M: A rapid boiling method for the preparation of bacterial plasmids. Analytical Biochemistry 198I, 124: 193-197. 8. National Committee for Clinical Laboratory Standards:
9. 10, 1I.
12.
Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. M7-T2. NCCLS, Villanova, PA, 1988. Sanders CC, Sanders WE:Type 1 I]-lactamases of gramnegative bacteria: interactions with IMactam antibiotics. Journal of Infectious Diseases 1986, t54: 792-800. Miller GH, Sabatelli FJ, Hare RS, Waltz JA: Survey of aminoglycoside resistance patterns. Developmental and Industrial Microbiology 1980, 21: 91-104. Ames GF: Resolution o[ bacterial proteins by polyaerylamide gel elcctrophoresis on slabs. Membrane, soluble, and periplasmic fractions. Journal of Biological Chemistry 1972, 249: 634-644. Hopkins JM, Towner KJ: Enhanced resistance to cefotaxime and imipenem associated with outer membrane protein alterations in Enterobacter aerogenes, Journal of Antimierobial Chemotherapy 1990, 25: 49-55.
Degranulation of Human Neutrophils after Exposure to Bacterial Phosphofipase C T.K. W a z n y 1, N. M u m m a w 1, B. Styrt 1'2. Because an endogenous phospholipase C (PLC) participates in neutrophil activation and because many bacterial pathogens produce PLCs, these studies examined the effect of PLC from Bacillus cereuson the release of the granule enzyme lysozyme from human neutrophils.Bacillus cereusPLC caused dose-dependent lysozyme release, and combined stimulation ofneutrophiis with PLC and fluoride led to increased secretion. Stimulation of neutrophil degranulation is a potential contributing factor for tissue damage in infections caused by PLC-producing organisms.
products released by bacteria, some of which resemble participants in the neutrophil's intrinsic activation pathway. For example, endogenous phospholipase C (PLC) is involved in neutrophil activation, and C-type phospholipases with varying substrate specificities are also produced and released as exoenzymes by many bacteria including Bacillus cereus (1). In the experiments reported here, we studied the effect of PLC from Bacillus cereus on human neutrophil degranulation. Materials and Methods. Histopaque 1077, two preparations of phospholipase C from Bacillus cereus (type III product no. P6315 and type XIII product no. P5527), Micrococcus lysodeikticus, lysozyme, sodium pyruvate, and NADH-Na 2 were obtained from Sigma Chemical, USA. Neutrophils were isolated from heparinized human blood as done in other studies (2) by Hypaque-Ficoll density gradient centrifugation, and contaminatingerythrocyteswere lysed with hypotonic NaCI solution. Trypan blue dye exclusion was used for evaluating viability of neutrophils. Three gi trypan blue (0.4 % solution in 0.9 % NaC1) was added to 3 gl of cell suspension and the percentage of cells stained by trypan blue was determined immediately by light microscopy. To measure degranulation, stimuli were added to 2 ml of cells in Hanks balanced salt solution, which were incubated in a shakingwater bath at37 °C for 30 min. The tubes were then vortexed briefly and a drop was removed for assessment of cell viability. One ml was removed from each tube, centrifuged at 200 g and 4 °C for 10 min,and the supernatant saved for enzyme assays. The residual 1 ml was lysed with 20 gl 10 % Triton X-100 for determination of total enzyme content. As a marker of degranulation we measured lysozyme, a component of both azurophil and specific granules, by lysis of Micrococcus lysodeikticus as described (2). Results represent lysozymereleased into the media as a percentage of the total lysozyme released by Triton, subtracting spontaneous release in the absence of stimuli. In selected experiments lactate dehydrogenase (LDH) was determined by measuring conversion of NADH into NAD during reduction of pyruvate into lactate. Results and Discussion. When lysozyme release was
Neutrophils kill microorganisms by ingesting them and releasing oxygen metabolites and lysosomal enzymes into the phagocytic vacuole. However, if lysosomal enzymes are released outside the cell, this can result in host tissue damage. Stimuliwhich might cause neutrophil degranulation in vivo include 1Department of Medicine, and 2Department of Microbiology and Public Health, B220 Life Sciences Building, Michigan State University, East Lansing, Michigan 48824-1317, USA.
measured as a marker of degranulation, exposure to two concentrations of PLC type III caused marked enzyme release with a dose response effect (Figure la). When we tested whether PLC directly influenced the lysozyme assay, adding a comparable amount of PLC to standard concentrations of lysozyme did not produce any significant changes in lysis of Micrococcus lysodeikticus. Therefore, we concluded that our Iysozyme measurements accurately indicated granule content release. To define whether PLC
Vol____~9, 1990
831
4O
¢
zo'
01i to PLC unitstml
0nn 5
10 15 20 mM NaF
Figure I: Percent of total lysozyme released after incubation with (a) 0.1 unil/ml type III PLC (n = 8) or 1 unit/ml type III PLC (n = 4), a n d (b) indicated concentrations of sodium fluoride (n = 2 or3 at each concentration).
DbO.h 20' a
~o
Figure 2" Percent of total lysozyme released in response to (a) 0.1 unit/ml type 111 PLC, 12.5 mM NaF or combined stimuli (n = 8), and (b) 1 unit/ml type XIII PLC, 12.5 mM NaF, or combined stimuli (n = 7).
affects neutrophil responses to other stimuli of degranulation, we used sodium fluoride. Lysozyme release after exposure to NaF alone was dose-dependent (Figure lb). However, the highest concentrations tended to cause damage of a large pereentage of ceils as measured by trypan blue staining. We selected concentrations of 12,5 mM for our experiments, in order to stimulate significant degranulation without killing the cells. Figure 2a demonstrates lysozyme released after exposure to PLC alone, NaF alone, and the combined stimuli. The release produced by combined stimuli was significantly higher (p < 0.05 by t-test) than the calculated sum of releases elicited by NaF alone and PLC alone. Experiments using 1 U/ml type XIII PLC showed additive but no synergistic activity in combination with fluoride (Figure 2b). PLC stimulated degranulation was very low in these experiments, possibly due to the use of the different PLC preparation. To determine whether lysozyme release in our experiments could be explained by cell death
instead of active secretion, we measured cytosol lactate dehydrogenase release and trypan blue dye exclusion. Lactate dehydrogenase proved to be an unreliable indicator of neutrophil death because of variable contribution from lysis of contaminating erythrocytes in the neutrophil preparation. Erythrocyte lysis was obvious as pink supernatants in some samples with substantial erythrocyte contamination, and it was not thought practicable to eliminate erythrocytes entirely from the neutrophil preparations because of the risk of leukocyte damage from more vigorous isolation procedures. Because of these problems we based our measurement of leukocyte viability on trypan blue staining which we have found to be comparable in sensitivity to L D H release in other studies (3). More than 95 % of these cells were viable after stimulation, when measured by this method. Therefore, we concluded that lysozyme release caused by PLCwas most likely due to secretion of granule contents from viable cells. To see whether additional lysozyme release could be achieved by even higher phospholipase concentrations, separate experiments were performed using a final concentration of 10 units per ml of Type III PLC. In these three experiments, PLC stimulated release of 71.6 + 1.7 % of total cellular lysozyme. Cells stimulated by PLC showed marked clumping on microscopic examination, making morphologic assessment difficult. A slightly higher percentage of ceils failed to exclude trypan blue, suggesting some cytotoxicity at the very high concentrations, but there was only 2.3 + 1.4 % excess cell death attributable to PLC. Addition of autologous plasma (10 % volume of approximately 1:1 plasma:PBS mixture from the cell separation) did not protect against either the degranulating effect or the cytotoxicity of PLC. Phospholipase C, an important enzyme in the turnover of phospholipids, appears to be an important endogenous mediator of neutrophil activation (4). Several studies suggest that exogenous PLC may also activate neutrophils. Most of the available studies concentrate on activation of the respiratory burst by bacterial phospholipases (5, 6), although phospholipase C obtained from Clostridium welchiiwas shown to activate both the respiratory burst and modest secretion of specific granule contents (7). Contradicting data showing reversible inhibition of the respiratory burst have also been reported (8). Since the respiratory burst and degranulation are frequently but not invariably linked, and the PLCs of Clostridium perfringens and Bacillus cereus may differ substantially in clinicat importance, we thought that investigation of each of these responses to Bacillus cereus PLC would be informative. Although
832
usually of low virulence, Bacillus cereus has shown potential for tissue invasion, cavitation and eschar formation in some settings (9, 10), suggesting that exotoxins might be of adjunctive importance in special situations. Results of our experiments, using phospholipase C from Bacillus cereus, demonstrated activation of neutrophils to release lysosomal contents and enhancement of the effects of a second stimulus. While it seems unlikely that PLC concentrations comparable to those causing degranulation would be achieved in circulation, we would expect that extremely high concentrations of bacterial exoproducts may be encountered by phagocytes in close contact with large numbers of bacteria at a focus of infection, so that these results have potential relevance to clinical situations. Although contributions from possible contaminants of the preparation cannot be altogether ruled out, neutrophil activation by other contaminating Bacillus cereus products could also be physiologically relevant. We did see degranulation in response to two different Bacillus cereus PLC preparations, suggesting that PLC is likely to be the agent producing the observed results. While in vivo confirmation has yet to be obtained, these findings suggest that release of neutrophil lysosomal enzymes may contribute to pathological changes caused by phospholipase-Cproducing organisms.
References 1. AvigadG: Microbialphospholipases.In: Bernheimer AW (ed): Mechanisms in microbial toxinology.John Wiley, New York, 1976,p. 99-161. 2. Styrt B, Johnson PC, Klempner MS: Differentiallysis of plasma membranes and granules of human neutrophils by digitonin. Tissue and Cell 1985, 17: 793-800. 3. Styrt B, Walker RD, White JC: Neutrophil oxidative metabolism after exposure to bacterial phospholipase C. Journal of Laboratory and Clinical Medicine 1989, 114: 51-57. 4. Verghese MW,Charles L, Jakoi L, Dillon SB, Snyderman R: Role of a guanine nucleotide regulatory protein in the activation of phospholipase C by different chemo-attractants. Journal of Immunology,1987, 138:4374-4380. 5. Patriarca P, Zatti M, Cramer R, Rossi F: Stimulation of the respiration of polymorphonuclear leucocytes by phospholipase C. Life Sciences 1970,9: 841.849. 6. Stevens DL, Mitten J, Henry 12."Effects of alpha and gamma toxins from Clostridium perffmgens on human polymorphonuclear leukocytes. Journal of Infectious Diseases 1987, 156: 324-333. 7. Grzeskowiak M, Bianea VD, DeTogni P, Papini E, Rossi F: Independence with respect to Ca2÷ changes of the neutrophil respiratory and secretory response to exogenous phospholipase C and possible involvement of diacylglyceroland protein kinase C. Biochimica et Biophysica Acta 1985,844: 81-90.
Eur. J. Clin. Microbiol. Infect Dis.
8. Gordon LL, Lee SN, Homan A, Prachand S, Weitzman SA: Phospholipase C reversibly inhibits respiratory burst activity in human neutrophils. Clinical Research 1987, 35: 850A. 9. Davey RT, Tauber WB: Posttraumatic endophthalmitis: the emergingrole of Bacillus cereus infection. Reviewsof Infectious Diseases 1987,9: 110-123. 10. SlimanR, Rehm S, Shlaes DM: Serious infectionscaused by Bacillus species. Medicine 1987,66: 218-223.
Inactivity of Terbinafine in a Rat Model of Pulmonary AspergUlosis H. J. Schmitt 1' 2,, j. A n d r a d e 1, F. E d w a r d s 1, Y. Niki 1, E. B e r n a r d 1, D. A r m s t r o n g 1 In a m o d e l of b r o n c h o p u l m o n a r y aspergillosis lerbinafine did not improve survival o f experimental animals in doses up to 80 m g/kg/day despite adequate lung concentrations. Pretreatment and aerosolization of the c o m p o u n d were also ineffective. Terbinafine was markedly less active in vitro when serum was used instead of Yeast-Nitrogen-Glucose-broth. It is concluded that a lack of bioavailability in the presence o f s e r u m m a y explain the lack of activity ofterbinafine in experimental aspergillosis.
Allylamines are a new class of agents which exhibit antifungal activity by inhibiting squalene epoxidase, a key enzyme in ergosterol biosynthesis of fungi (1-4). In vitro, antifungal activity of these agents has been demonstrated against a broad spectrum of fungi (5-7). We previously found that terbinafine was highly active against clinical isolates of A s pergillus spp. (8). Since the options for treatment of patients with pulmonary aspergiilosis are not very satisfactory in terms of efficacy and toxicity (9, 10), we evaluated terbinafine in a standardized model of bronchopulmonary aspergillosis. Materials and Methods. Terbinafine was a gift from
Sandoz, Switzerland. For treatment of experimental animals, terbinafine was initially dissolved in dimethyl sulfoxide (DMSO) and further dilutions were made in sterile saline just prior to intraperitoneal (i.p.) administration. D M S O (5 %) and 0.02 % iInfectious Diseases Service, Department of Medicine and Microbiology Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. 2Children's Hospital, Johannes Gutenberg University, Langenbeckstral3e 1, 6500 Mainz, FRG.
