Synergism of Candida albicans and delta toxin producing staphylococcus aureus on mouse mortality and morbidity: Protection by indomethacin

Zbl. Bakt. Hyg. A 269, 377-386 (1988)

Synergism of Candida albicans and Delta Toxin Producing Staphylococcus aureus on Mouse Mortality and Morbidity: Protection by Indomethacin EUNICE C. CARLSON Michigan Technological University, Houghton, Michigan 49931, USA

Received February 11, 1988 . Accepted June 6, 1988

Abstract Twelve Staphylococcus aureus strains, six positive and six negative for b-toxin production, were studied for synergistic effects on mouse mortality and morbidity when combined with Candida albicans and inoculated intraperitoneally (i.p.). S. aureus strains producin b-toxin were found to exhibit a relatively great synergistic decrease (between near 103- 10 fold) in LDso (dose necessary to kill 50 % of exposed animals in five days) when combined with a nonlethal dose of C. albicans and injected i. p. S. aureus strains which did not produce b showed less of a synergistic effect with C. albicans (10-10 2-fold drop in LDso). A synergistic effect on mortality could also be produced when animals were dually injected with C. albicans and sterile growth filtrates from the b-toxin producing strains or the pur ified b-toxin . The lethal agent in the culture filtrate was, like b-toxin, sensitive to lecithin and insensitive to heat. Indomethacin protected animals from the C. albicans-filtrate induced death. Blood measurements made following i. p. injection of b-toxin and C. albicans revealed chemistry changes indicative of shock, kidney and liver damage; o-roxin alone caused no significant chemistry changes whereas C. albicans alone caused some blood chemistry changes but liver and kidney damage was not indicated. No synergism on mortality was found between C. albicans and purified a-toxin or toxic shock syndrome toxin-L

r

Zusammenfassung Zw6lf Staphylococcus aureus-Stiimme, von denen sechs fur b-Toxin-Bildung positiv und sechs negativ waren, wurden auf synergistische Wirkung auf die murine Mortalitat und Morbiditat hei Kombination mit Candida albicans und i. p. Verimpfung untersucht. S. aureus-Stamme, die b-Toxin bildeten, zeigten eine relativ hohe synergistische Abnahme (erwa urn das 103_ his lOs-fache) in der LDso (erforderliche Dosis zur Torung von 50% der exponierten Tiere in 5 Tagen) hei Kombination mit einer nicht leralen Dosis von C. albicans und i. p. Injektion. S. aureus-Stamme ohne b-Toxin-Bildung zeigten einen geringeren synergistischen Effekt mit C. albicans (Verminderung der LDso urn das 10- his 102-fache). Eine synergistische Wirkung auf die Mortalitat konnte ebenfalls erzeugt werden, wenn die Tiere zweifach mit C. albicans und sterilen Wachstumsfiltraten von den b·Toxin bildenden Stammen oder dem gereinigten b-Toxin beimpft wurden. Das letale Agens im Kulrurfiltrat war

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wie b-Toxin lecithinempfindlich und hitzeunempfindlich. Indomethacin schiitzte die Tiere gegen den durch das C. albicans-Filtrat induzierten Tod. Blutuntersuchungen nach i. p, Injection von b-Toxin und C. albicans ergaben chemische Veranderungen, die auf Schock sowie Nieren- und Leberschadigung hinwiesen. b-Toxin allein erzeugte keine signifikanten chemischen Veranderungen, wogegen C. albicans allein einige chemische Veranderungen im Blut hervorrief, Leber- und Nierenschadigung aber nicht angezeigt wurde. Bei der Mortalitat wurde kein Synergismus zwischen C. albicans und gereinigtem a-Toxin oder ToxicShock-Syndrom-Toxin 1 festgestellt.

Introduction In previous reports from this laboratory, we described enhancement by C. albicans of S. aureus, Serratia marcescens, and Streptococcus faecalis in the establishment of experimental infection in mice when fungi and bacteria were inoculated intraperitoneally (i. p.) (6, 8). It was found that the degree of mortality resulting from the C. albicans-S. aureus infections varied greatly according to bacterial strain and, therefore, may be dependent on the array of toxins produced by the bacteria in the host (7). The study reported here employed S. aureus strains which were characterized for their hemolytic toxins, a, ~, y, and b, to examine the role of these toxins in the synergism between C. albicans and S. aureus on mouse mortality. In addition, purified a-toxin, btoxin and toxic shock syndrome toxin-1 (TSST-1) (4,26) were examined for possible synergism with the fungus.

Materials and Methods

Mice. Outbred CD-1 mice were obtained from Charles River Laboratories, Wilmington, MA, USA. Male mice weighing between 22 and 25 g were used with inoculated mice caged in groups of four. Pathogens. C. albicans L-1 was from the microbiology laboratory stock culture at Michigan Technological University, Houghton, MI, USA. This strain has been used in previous publications (6, 7, 8) and has an i. p. LDso in mice of 3.2 x 10 8 colony forming units (CFU) at the time of this study. The S. aureus strains designated were obtained from the following sources: FRI-1169 and FRI-1214 from M. S. Bergdoll (University of Wisconsin, Madison, WI, USA); 689, PG-114, and 303 from F. A. Kapral (Ohio State University, Columbus, OH, USA); Todd-555 and 869 from J. Todd (Denver Children's Hospital, Denver, CO, USA); Cowan from G. Best (University of Georgia, Augusta, USA); 25904 from the American Type Culture Collection (Rockville, MD, USA); PG-23 from Procter and Gamble Co, (Cincinnati, OH, USA); and CDC-19 and CDC-II from A. Reingold (Centers for Disease Control, Atlanta, GA, USA).S. aureus strains are listed in Table 1 along with TSST-1 and a, ~, y and b-toxin production. Culture Filtrates. Culture filtrates were produced by inoculating 0.1 ml of a 15 h tube culture (Todd-Hewitt broth, Difco Laboratories, Detroit, MI, USA) into 25 ml of the same medium in a 250 ml flask and incubated at 37°C for 24 h with aeration (Environmental shaker G-24 New Brunswick Scientific Co., Edison, NJ, USA) at 220 rpm, removal of the cells by centrifugation (5,000 x g) at 4°C and sterilization of the resulting supernatant by filtration [Nalgene Model 120-0020 with 0.2 11m pore size (Nalge Co., Rochester, NY, USA)]. Sterility of the resulting culture filtrate was confirmed by swabbing onto mannitol salt agar (Difco Laboratories). Filtrates were maintained at 4°C until immediately before use and used the same day of preparation. In some instances the biological activity of the

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filtrate or O-toxin was tested after heating at 65°C at dose concentration for one h before inoculation into animals. The heat insensitive hemolytic units (HU) of I)-toxin/ml in the culture filtrates were determined on human red cells (RBCs) by the tube technique of Bernheimer and Schwartz (3) after heating samples at 65°C for one h (19, 33). In certain filtrates the O-toxin activity was eliminated by the addition of 100 ug lecithin/ml (17) (Sigma Chemical Co., St. Louis, MO, USA). A HU was defined as the dilution of culture filtrate required to release 50% of the RBC hemoglobin. Estimates of O-toxin in J-tg/ml in filtrates were made by determining the heat resistant HU/ml and converting to J-tg/ml by the use of a purified O-toxin standard solution with HU/ml measured under identical conditions. Production of TSST-l and Hemolytic Toxins. Production by S. aureus strains of TSST-1 was determined by immunodiffusion tests under the direction of J. J. Kirkland (24) (Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, OH, USA) by methods previously described. Two samples of purified TSST-1 were gifts from R. Stone and M. S. Bergdoll prepared by described methods (23). Biological activity of TSST-1 samples was demonstrated by lethality in rabbits. Purified O-toxin preparations was the generous gift of Dr. F. A. Kapral (18) (Ohio State University) with method of purification described previously. Purified a-toxin was purchased from Connaught Laboratories (Toronto, Ontario, Canada). The production by S. aureus strains of a, ~, and I)-toxins, was qualitatively determined by the agar immunodiffusion method of Elek and Levy (12) employing staphylococcal antitoxin (Connaught Laboratories). Strains found to be O-toxin negative by the immunodiffusion test also failed to exhibit a synergistic hemolytic effect with a ~-toxin producer (S. aureus 303) on sheep blood agar by the described method (13), thus confirming the lack of O-toxin production. Strains of S. aureus positive for a could not be characterized for y by the tube or agar immunodiffusion tests because y is masked by a and inhibited by agar (14, 33). Strains negative for a where characterized for y by the tube test as described previously (9) and by the Elek-Levy immunodiffusion test with agarose (Bio-Rad Laboratories, Richmond, CA, USA; ultra pure grade) substituted for agar (9). Strains positive for a were tested for y production by analytical isoelectric focusing with overlay containing thrice-washed rabbit RBCs (33) where y-hemolysis is clearly observed close to the cathode in the pH range of 9-9.5 (10). Animal inoculations. Cultures used for inoculation were grown on sheep blood agar (for S. aureus) or on Sabouraud dextrose agar (for C. albicans), washed, virgorously mixed, and titered as described previously (6). Organisms or toxins (TSST-1, 0 or a-toxin) were introduced i. p., with each desired dose suspended in 0.2 ml of nonpyrogenic saline (Abbott Laboratories, Chicago, IL, USA) and mixed immediately before injection. When only one agent was used, 0.2 ml of saline was substituted for the second agent. Intravenous (i.v.) inoculations were made into the tail vein. Organisms and filtrates were introduced i. p.; C. albicans was suspended in 0.2 ml of nonpyrogenic saline and mixed immediately before injection with filtrate. When only filtrate was used, 0.2 ml of saline was substituted for the C. albicans. When only C. albicans was inoculated, Todd-Hewitt broth was added to make all volumes injected per experiment, constant. In some experiments animals were protected from the C. albicans-S. aureus culture filtrates by indomethacin (Sigma Chemical Co.) or lecithin (Sigma Chemical Co.). This was done as follows. Indomethacin was dissolved in a small volume of ethanol and diluted to the required concentration with nonpyrogenic saline. Animals protected with indomethacin were injected 15 minutes before pathogen filtrate challenge at a dose of 100 ug in 0.2 ml i. p. and continued twice daily for three days. Thereafter 50 ug were given daily. Nonprotected animals were given injections lacking indomethacin but otherwise identical. When lecithin was used to neutralize O-toxin in culture filtrates, lecithin was first dissolved in a small volume of ethanol and diluted into the culture filtrate at a final concentration of 100 ug per ml filtrate. Animals not protected with lecithin received filtrates treated identically but lacking lecithin. Injected animals were observed five days for mortality. All experiments were repeated and were reproduceabile.

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Clinical Pathology. Blood was drawn by cardiac puncture from mice anesthetized with CO 2 , One m1 was transferred to a tube (serum separator 5960, Becton Deckinson [BD], Rutherford, N], USA) for blood chemistry analysis and 0.3 ml was transferred to a whole blood collector with EDTA (BD-5961).Blood was chemicallyanalyzed at Roche Biomedical Laboratories, Columbus, OH, and hematology was done by Procter and Gamble, Miami Valley Laboratory under the direction of K. Stretzel. Statistical analysis. The i. p. LDso was determined by the moving average method (2) for each strain of S. aureus alone and as influenced by a nonlethal dose (1/3 LDso or 108 CFU)of C. albicans as described previously (7). Groups of six or more animals were given doses of each S. aureus strain to determine at least one dose which resulted in no mortality, one dose giving complete mortality, and two doses yielding partial mortality. Where no dose tested yielded a non-mortality group, probit analysis was employed to determine the LDso (15). Additional data were analyzed by the Fisher exact test and the method of least significant difference (1).

Results Strains of S. aureus used, production of TSST-1 and a, ~, y, (I)-toxin are given in Table 1. Also in Table 1 the size of the LDso dose of the strains of S. aureus as influenced by a nonlethal dose of C. albicans is presented. It can be seen (Table 1) that the dose of (I)-positive strains. when introduced with C. albicans, could be reduced by near three to five orders of magnitude before its ability to kill half of the exposed mice i. p. was lost whereas the LDso of (I)-negative strains was reduced by about two orders of magnitude or less by the presence of C. albicans. The amount of C. albicans necessary for the above synergistic effect on mortality was determined by inoculating groups of six animals with 10 8 CFU S. aureus FRI-1169 and varying amounts of fungus. Under these conditions the LDso of C. albicans was 5.2 7 X 10 CFU or about 1/6 its LDso alone. To determine if C. albicans was acting synergistically with (I)-toxin, as indicated by the results presented inTable 1, various amounts of purified (I)-toxin were combined with 10 8 CFU C. albicans and injected i. p. into groups of mice. Table 2 shows that dual inoculation with the toxin and fungus together, at doses which "separately caused no animal deaths, resulted in 100% mortality. It is further shown that C. albicans lowered the lethal dose of (I)-toxin by about one order of magnitude. The amount of C. albicans necessary for the synergistic reaction with (I)-toxin was measured by dually inoculating groups of six animals i. p. with 500 ug of (I)-toxin and various amounts of fungus. The LDso for the fungus when combined with (I)-toxin was 1.2 X 10 7 CFU or near 1/24 the LD so of the fungus alone. C. albicans was also tested by the i. p. route for effect on mouse morbidity and mortality with a-toxin. Using 1/2 the dose (or 10 hemolytic units) of a-toxin which resulted in some mortality when used alone, in combination with the fungus, no synergistic effect on morbidity or mortality was observed. TSST-1 from two laboratories was also tested for effect on mouse morbidity and mortality with and without C. albicans. No death resulted in the groups of six animals inoculated with 1, 10 and 100 ug of TSST-1 i. p. or 10 ug i. v. alone or in combination with an i. p, inoculation of 108 CFU C. albicans. The effect on mouse mortality of i. p. inoculations of sterile 24 h culture filtrates of representative S. aureus strains alone and in combination with 0.5 X 10 7 CFU C.

Synergism of Ca. albicans and Delta Toxin Producing S. aureus

381

Table 1. Synergism of C. albicans and various strains of S. aureus on mouse mortality" LDso S. aureus alone Strain

Hemolytic Toxin

689

<1

7.0 x 109 2.2 x 109

~3

303

~

1.2 x 1010 6.0 x 109

~2

ATCC-25904

y

2.8 x 109 5.0 x 108

-50

869

b

5.3 x 109 7.0 x 106

-750

Cowen

<1,y

5.0 x 109 1.3 x 108

-40

CDC-19

<1,y

+

4.5 X 109 1.2 x 108

~40

PG-23

b,y

+

2.9 X 109 3.5 x 107

-800

Todd-555

b,y

+

2.2 X 109 9.0 x 106

-2,500

FRI-1169

b,

+

1.5 2.5

PG-1l4

e.s, y

8.5 X 109 1.0 x 106

-8,500

CDC-ll

<1,~,b,y

2.1 x 109 1.1 x 105

-20,000

FRI-1214

<1, ~,b

~,y

TSST-1

+

LDso S. aureus

X X

+ C. albicans

1010 ~60,000b 105

9

1.8 x 104 ~200000 1.0 x 10 ,

Groups of animals were injected i.p. with 108 CFU C. albicans along with various doses of S. aureus so as to determine an LDso of the S. aureus as influenced by the C. albicans during a five day observation period. b The synergism between these two organisms has been reported (7) with the results here in good agreement with those previously reported. a

albicans is presented in Table 3. Filtrates from strains which produce 6-toxin showed a synergistic effect with C. albicans on mouse mortality while the one tested which lacked 6-toxin did not. It is also seen in Table 3 that the biological activity of the S. aureus FRI-1169 filtrate, like 6-toxin, was inactivated by lecithin but not heat. The heated S. aureus FRI-1169 filtrate contained 320 IJ.g 6-toxin/ml. When lecithin was added to the S. aureus FRI-1169 culture filtrate 6-toxin activity was no longer detectable at the lowest dilution tested (6 < than 100 IJ.g/ml). Indomethacin injected separately during the test period protected animals from the C. albicans-S, aureus FRI-1169 culture filtrate-induced death.

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E. C. Carlson

Table 2. Synergism of C. albicans and O-toxin from S. aureus on mouse mortality Dose ~-toxin (mg)

C. albicans (CFU)

Dead Miceffotal

1.0 0.5 b 0.2 0.1 2.0 1.0 0.5 0.2 a b

6/6' 5/6' 5/6' 0/6 6/6 0/6 0/6 0/6

Significance difference from animal group inoculated i.p. with the same amount of 0toxin in the absence of fungus (P < .05 by Fisher exact test). This O-toxin-C. albicans dose was repeated on an additional six animals after first heating ~-toxin at dose concentration at 65°C for 1 h. The same results were obtained.

Table 3. Synergism of S. aureus growth filtrates and C. albicans on mortality in mice. Protection by indomethacin and lecithin

Experiment

Culture Filtrate (S. aureus strain)

Dead Miceffotal (S. aureus Filtrate

A

1. CDC-19 (o-negative)

0/6

B

1. FRI-1169 (o-positive) 2. PG-23 (o-positive)

11/12 b 6/6 c

C

1. FRI-1169 2. FRI-1169

4/12 d (lecithin)" 12/12

D

1. FRI-1169 2. FRI-1169

3/12 d (indomethacin)' 12/12

a

b

c d

e f

+ C. albicansi'

Dual dose consisted of 1 ml culture filtrate containing 5 X 10 7 CFU C. albicans. Animals (n = 6) were not killed by 1 ml broth containing 5 X 10 7 CFU C. albicans or 1 ml culture filtrate from S. aureus FRI-1169, CDC-19 or PG-23 alone, during the five day observation period. Significant difference (P < .01 by Fisher exact test) from animal group inoculated i. p. with filtrate or C. albicans alone. This experiment was repeated after heating the filtrate for 1 h at 65°C with similar results (8 of the 12 treated animals died). Significant difference (P < .01 by Fisher exact test) from animal group inoculated i. p. with filtrate or C. albicans alone. Significant (P < .01 by Fisher exact test) from animal group inoculated (i.p.) with the identical S. aureus FRI-1169 filtrate and C. albicans dose. Lecithin. Filtrate was first treated with 100 ug lecithin/ml to inactivate o-toxin(15). Indomethacin. Mice treated with 100 J.tg indomethacin (i. p.) 1/2 h before filtrate - C. albicans injection with additional treatment of indomethacin as described in the Materials and Methods section.

Synergism of Ca. albicans and Delta Toxin Producing S. aureus

383

Table 4. Blood chemistrychangesin mice24 h followingan J.P. injectionof C. albicans'and b-toxinb from S. aureus Blood Urea

Treatment Group RBC (n = 6) Saline

Nitrogen

(x 106/ml) (mg/dL) 9.7±o.3

21±2

Calcium SGPT (mg/dL) 9.4 ± 0.3

(lUlL) 98±21

Hematocrit Glucose (%)

(mg/dL)

41.2±0.8

24S±30

C. albicans & b-toxin

11.8 ±0.6c 161± ll d

7.S±0.3d 398±111 d 46.7±2.0 d

C. albicans

10.7±0.l c

17±2

9.S±2.8

b-toxin

1O.4±0.1

19±1

9:9± 1.9 144±24

a b c

d

20±6

70±2C

44.0±0.9

68±3 c

41.0 ± O.S

266± 14

C. albicans dose contained S X 107 CFU/mouse. 6-toxin dose contained SOO ug/mouse, Significantly different from saline control by p < .01 by the method of least significant difference. Values represent x ± S.E. Significantly different from saline control by < .001 by the method of least significant difference.

Table 4 summarizes changes in blood chemistry and hematology in animals receiving b-toxin, C. albicans, or b-toxin and C. albicans combined. It is noted that the combination of agents induced changes indicative of liver and kidney damage, and shock whereas b-toxin alone had no detectable effect and C. albicans alone effected the parameters tested to a lesser degree or not at all.

Discussion A number of reports indicate that C. albicans supports the growth of S. aureus. In vivo, the proteolytic products resulting from growth of C. albicans are able to convert a serum-protein medium unsuitable for the growth of S. aureus to suitable for S. aureus as well as other bacterial genera (28). In our laboratory we found an enhancement of S. aureus and various other bacteria in the establishment of experimental mouse infections when the bacteria were inoculated i. p. with C. albicans (4). In addition, S. aureus has been found to be a frequent opportunist in experimentally induced candidiasis (29). Clinically antifungal agents alone have been reported to cure chronic combined S. aureus and C. albicans infections (27). In addition to a general effect where C. albicans protects various bacteria, it has also been previously found that certain S. aureus strains were much more virulent in mice when introduced with C. albicans than others suggesting that certain toxic products of S. aureus and C. albicans acted synergistically (S). The experiments reported here demonstrate a synergistic effect between C. albicans and b-toxin producing S. aureus, S. aureus culture filtrates containing b-toxin, or purified b-toxin resulting in morbidity and mortality in mice. The strains which produced a, Por y-toxin in the absence of other hemolytic toxins were relatively nonlethal to mice when introduced with C. albicans. In as much as the most actively synergistic strains produce b, it is reasonable that the synergism found

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E. C. Carlson

with the purified a-toxin is at least in part responsible for the effect of C. albicans and S. aureus observed on mouse mortality. That the synergism between C. albicans and S. aureus FRI-1169 culture filtrate was due to O-toxinwas strongly implied by the finding that the biological activity of the culture filtrate was, like O-toxin(17, 20), destroyed by lecithin but not by heat. Although most O-toxin positive strains in this study also produced TSST-l, C. albicans displayed no synergism with TSST-l on mouse mortality. We found that a greater dose of fungus is necessary to demonstrate synergism with living S. aureus than with a-toxin. This is reasonable in that bacteria injected i. p. into mice will not reproduce unless introduced with the fungus, which somehow protects and allows bacterial reproduction, and presumably toxin production (6, 8). When purified a-toxin is introduced no bacterial growth is necessary for its production and it is possible that a dose of fungi, smaller than that needed for protection, is required for the synergism of the fungus with the toxin. A question arises as to why the addition of C. albicans decreases the LDso of o-toxin 10 fold whereas C. albicans decreases the LDso of live, a-producing S. aureus by three or more orders of magnitude. The vivo synergism cannot be expected to quantitatively reproduce the synergism observed with C. albicans and a boulous dose of toxin as toxin could be produced continuously in the vivo model. In addition, other toxins may well be produced by S. aureus which interact with C. albicans. In this study the S. aureus cultures were produced with aeration but without added COz [COz has not been found to enhance O-toxin production (34)]. When S. aureus is cultured in the presence of COz, additional toxins, which well could also be produced in vivo, such as y (9) and protease (31) are detected. As production of various toxins is dependent on culture conditions conclusions about production in the vivo model from the vitro culture cannot be made. However, production of O-toxin in intraperitoneal abscesses by S. aureus has been reported (N. Chamberlain and F. Kapral, Abstr. Annu. Meet. Am. Soc. Microbiol. 1985, B 209, p. 53). A recent publication (22) describing a synergistic effect between C. albicans and a protease of Pseudomonas aeriginosa on mortality in burned mice supports the possibility that additional staphylococcal toxins may be involved synergistically with C. albicans in the vivo model. A great deal of variation in effectiveness of synergism with C. albicans among the 6-producing S. aureus was observed. One could speculate that the 800 fold increase in mortality due to the combination of c. albicans with S. aureus 869 was due mainly to the O-toxin while the 200,000 fold mortality increase resulting the C. albicans-S, aureus FRI-1214 combination was due to a-toxin in combination with an additional S. aureus toxin(s). Visual observation of single colonies on sheep blood agar revealed that S. aureus FRI1214 was the strongest ~-toxin producer in this study; ~-toxin, known to act synergistically with O-toxin (31), could further amplify the C. albicans-b-toxux synergism. The contribution of important candidal virulence factors, such as proteases (25), in this synergism has yet to be assessed. It must be emphasized that the study reported here was not designed as a model for human disease, but rather to study further the fungal-bacterial synergistic effect previously reported. However, the finding that the fungus interacts with o-toxin is noteworthy in that this toxin has been reported to stimulate synthesis of PGEz and of other prostaglandins in fibroblasts (11), act as an anterotoxin and increase the cyclic AMP content of the guinea pig ileum (19). Protection of animals from candidal-S. aureus filtrate by indomethacin supports a prostaglandin mediated death by this synergism (5). Zymosan, the cell wall material of C. albicans has been reported to enhance

Synergism of Ca. albicans and Delta Toxin Producing S. aureus

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production by macrophages of PGE2 (16) and it is reasonable that if both pathogens are contributing to abnormal prostaglandin synthesis a synergistic relationship could result. Indeed, a prostaglandin involvement could explain the shock type of symptoms reported in this study, some of the many diverse symptoms found in patients with chronic candidosis (31) and it has also been suggested as possibl y playing a role in staphylococcal infections (30).

Acknowledgments. This research was supported by a grant from the Procter and Gamble Company and the American Heart Association of Michigan. Jane Stevens is greatfully acknowledged for manuscript preparation.

References

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Prof. Dr. Eunice C. Carlson, Michigan Technological University, College of Sciencesand Arts, Dept. of Biological Sciences, Houghton, Michigan 49931, USA