New Model Of Oropharyngeal and Gastrointestinal Colonization by ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 1996, p. 1604–1609 0066-4804/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 40, No. 7

New Model Of Oropharyngeal and Gastrointestinal Colonization by Candida albicans in CD41 T-Cell-Deficient Mice for Evaluation of Antifungal Agents AMY M. FLATTERY,* GEORGE K. ABRUZZO, CHARLES J. GILL, JEFFREY G. SMITH, AND KEN BARTIZAL Antibiotic Discovery and Development, Merck Research Laboratories, Rahway, New Jersey 07065-0900 Received 11 December 1995/Returned for modification 14 February 1996/Accepted 14 April 1996

A new model for the evaluation of antifungal compounds against oropharyngeal and gastrointestinal mucosal colonization by Candida albicans was developed. To simulate the immune deficiency observed in AIDS patients, mice were depleted of CD41 T lymphocytes by the injection of either GK1.5 hybridoma cells or purified anti-CD41 monoclonal antibody derived from GK1.5 hybridoma cells in tissue culture. Fluorescenceactivated cell sorter analysis of splenic lymphocytes confirmed the elimination of the CD41 T-cell population. Gentamicin, a broad-spectrum, nonabsorbable aminoglycoside antibiotic, was given via the drinking water to reduce the normal gastrointestinal microflora, allowing less competition for colonization of the gastrointestinal tract by the C. albicans isolates. Mice were challenged by gavage and swabbing their oral mucosae with a pure culture of C. albicans. Gentamicin was withdrawn 3 days postchallenge, and antifungal compounds were administered via the drinking water ad libitum at concentrations ranging from 25 to 400 mg/ml. L-693989, a water-soluble phosphorylated cyclic lipopeptide prodrug of pneumocandin Bo, and L-733560, a semisynthetic derivative of pneumocandin Bo, are inhibitors of 1,3-b-D-glucan synthesis that exhibit potent in vivo antiCandida spp. and anti-Pneumocystis carinii activities. The efficacies of L-693989, L-733560, fluconazole, ketoconazole, and nystatin were evaluated in this new oropharyngeal and gastrointestinal model of mucosal colonization. L-693989, L-733560, fluconazole, and ketoconazole showed superior efficacies in reducing the numbers of C. albicans CFU per gram of feces and the numbers of oral CFU relative to those in sham-treated controls in this model, while nystatin was moderately effective in reducing oral and fecal colonization by C. albicans in this model.

Although Candida albicans is present in many mammals including humans, normal bacterial flora and various immune factors usually restrict the growth of C. albicans in the alimentary tracts of immune competent hosts (1, 29, 33). Infection of the alimentary tract mucosae, including the mucosae of the oropharynx, esophagus, and gastrointestinal tract, with C. albicans is occurring with greater frequency, presumably because of the increased population of immune compromised individuals (5, 13, 15, 16, 21, 24, 30, 35, 37). Recent evidence suggests that cell-mediated immunity, and more specifically, CD41 T lymphocytes, play an important role in resistance to mucosal candidiasis (2, 3, 6–8, 10, 26, 30, 33). Patient populations with AIDS or other defects in cellular immune function show an increased incidence of mucocutaneous, but not necessarily disseminated, candidiasis (13, 15, 21, 29, 30), whereas patients with phagocytic cell defects, such as those that occur in patients with neutropenic or chronic granulomatous disease states, show a higher incidence of disseminated candidiasis (5, 10, 23, 25, 33). A combination of defective cell-mediated immunity and phagocytic cell defects in athymic beige (bg/bg nu/nu) mice was found to predispose them to severe mucosal candidiasis with subsequent Candida dissemination (6). Euthymic beige (bg/bg nu/1) mice selectively depleted of CD41 T lymphocytes had increased susceptibility to mucosal candidiasis without dissem-

ination of infection (8). Existing mouse models of mucosal candidiasis use combinations of chemically induced immune suppression, elimination or alteration of the host microflora by administration of antibiotics, high inocula, trauma, infant animals, or animals with congenital, functional, physiological, immunological, or metabolic defects to facilitate colonization of the gastrointestinal tract by C. albicans (9, 11, 12, 14, 17–20, 28, 31, 34). We have developed a murine model of chronic oropharyngeal and gastrointestinal mucosal colonization by C. albicans specifically for the evaluation of antifungal agents. This model uses a combination of selective CD41 T-cell depletion in complement component 5-deficient DBA/2 mice to initiate a specific immune deficiency and reduction of the normal gastrointestinal microflora with antibiotics. In this model, DBA/2 mice were selectively depleted of CD41 T lymphocytes by treatment with a rat immunoglobulin G2 monoclonal antibody (MAb) secreted by GK1.5 hybridoma cells, which is specific for mouse CD41 T cells. The activities of the commercially available antifungal agents fluconazole, ketoconazole, and nystatin and the lipopeptide antifungal agents L-693989 and L-733560 were evaluated in this model of mucosal colonization. MATERIALS AND METHODS Microorganism. C. albicans MY1055 (Merck Culture Collection) was maintained by monthly transfers on Sabouraud dextrose agar plates (SDA; BBL, Cockeysville, Md.). Growth from an 18- to 24-h SDA culture of C. albicans MY1055 was suspended in sterile saline to a concentration of 108 cells per ml, as determined by hemacytometer counts and verified by plate counts. Mice were challenged with 0.2 ml of the yeast suspension by gavage (2 3 107 cells per mouse) and additionally by swabbing their oral cavities with the yeast suspension while gently abrading the buccal mucosa by rotation of the swab.

* Corresponding author. Mailing address: Antibiotic Discovery and Development (R80T-100), Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065-0900. Phone: (908) 594-5258. Fax: (908) 5945700. 1604

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TABLE 1. Percent CD41, CD81, and Thy-1.21 splenic T lymphocytes in DBA/2 mice over time % of the indicated cells after the following treatmenta Day

GK1.5 hybridoma CD4

7 14 21 28 Untreated control

1

0.1 6 0.0b 0.2 6 0.0b 0.1 6 0.0b 7.5 6 4.5b 21.2 6 1.1

Anti-CD4 MAb

1

1

CD8

1

Thy-1.2

16.8 6 4.0 12.3 6 1.3 12.7 6 2.7 14.1 6 2.4

CD4

19.9 6 4.8 12.7 6 1.5b 13.5 6 2.4b 18.6 6 2.6

8.6 6 0.8

0.1 6 0.0b 1.1 6 1.0b 2.4 6 0.5b 5.7 6 0.2b

CD81

Thy-1.21

13.0 6 1.0b 10.8 6 2.2 8.6 6 0.9 7.8 6 0.9

12.3 6 0.8b 12.0 6 1.3b 10.3 6 0.8b 12.8 6 1.3b

27.6 6 3.6

DBA/2 mice (n 5 three per time point) were injected with 9 3 10 GK1.5 hybridoma cells on day 0 or three injections of purified anti-CD4 MAb: 300 mg on day 0, 200 mg on day 4, and 100 mg on day 7. Values are means 6 standard errors of the means, with the values for untreated controls averaged over all time points. b Significantly different from controls (P , 0.05). a

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Cell culture and ascites production. GK1.5 hybridoma cells (ATCC TIB 207) were cultured in high-glucose (5 g/liter) Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, Mo.) with 10% fetal bovine serum (Sigma), 1% L-glutamine (Sigma), 100 U of penicillin (Sigma) per ml, and 100 mg of streptomycin (Sigma) per ml at 378C under 5% CO2. Cells for injection were passaged and incubated for log-arithmic-phase growth. Cultures were harvested and centrifuged at 400 3 g for 8 min to pellet the cells. Cells were washed twice with the medium described above, counted on a hemacytometer, and determined to be greater than 95% viable by trypan blue dye exclusion. Outbred athymic, nu/nu, Swiss Webster mice (Taconic, Germantown, N.Y.) were used for ascites production of anti-CD41 MAb. Mice were primed by intraperitoneal (i.p.) injection with 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane; Sigma). Ten days after pristane priming, mice were injected i.p. with 5 3 106 GK1.5 hybridoma cells. Ascites was collected from the mice, centrifuged at 400 3 g for 10 min to remove cellular debris, and then stored frozen at 2208C until it was used for antibody purification. MAb was purified from ascites by passage over a protein G column (MabTrap G; Pharmacia LKB, Piscataway, N.J.) and was quantitated by a protein assay (Bio-Rad, Rockville Center, N.Y.) with a bovine gamma globulin standard. Mice. Female DBA/2N mice (average weight, 19 to 21 g; Taconic) congenitally immune deficient in complement component 5 were used in the studies. Gentamicin (Garamycin injectable; Schering, Kenilworth, N.J.), a nonabsorbable broad-spectrum antibacterial agent, was administered at 0.1 mg/ml in the drinking water from 4 days prior to C. albicans challenge through 3 days postchallenge. Each mouse was injected either with 9 3 106 GK1.5 hybridoma cells subcutaneously 4 days prior to challenge or with three i.p. injections of 300 mg of purified MAb in sterile saline, administered 3 days prior to, on the day of, and 1 week after challenge. All procedures with animals were performed in accordance with the highest standards for the humane handling, care, and treatment of research animals and were preapproved by the Merck Institutional Animal Care and Use Committee. The care and use of research animals at Merck meets or exceeds all applicable local, national, and international laws and regulations. FACS analysis. CD41, CD81, and Thy-1.21 T-cell counts were analyzed by fluorescence-activated cell sorter (FACS) analysis. Spleens were aseptically removed, and by using frosted glass microscope slides, splenic tissue was teased apart to create a cell suspension. The cells were resuspended in 3 ml of phosphate-buffered saline (PBS; GIBCO, Grand Island, N.Y.) and were centrifuged at 400 3 g for 5 min. The supernatant was decanted, and the cell pellet was resuspended in 1 ml of ACK lysing buffer (GIBCO), vortexed for 1 min to lyse the erythrocytes, and then diluted in 3 ml of PBS and centrifuged at 400 3 g for 5 min. The supernatant was decanted and the cells were resuspended in 4 ml of PBS. Then, 100 ml of the spleen cell suspension was incubated with rat antimouse MAb at a concentration of 5 mg/ml for 30 min at room temperature. The cells were stained either with fluorescein isothiocyanate (FITC)-conjugated L3T4 (CD4) (PharMingen, San Diego, Calif.) and R-phycoerythrin (PE)-conjugated Ly-2 (CD8a) (PharMingen) or with FITC-conjugated Thy-1.2 (PharMingen), which reacts with 100% of the T cells in mice expressing the Thy-1.2 allele. The cells were washed with 3 ml of PBS and were centrifuged at 400 3 g for 5 min. The supernatant was decanted, and the cell pellet was resuspended in 200 ml of propidium iodide (Sigma) at 1 mg/ml. Samples were run on a FACScan analyzer (Becton Dickinson, San Jose, Calif.). Enumeration of viable C. albicans isolates. Mice were surveyed for the presence of C. albicans isolates in the oral cavity and feces on days 0, 3, 5, 10, and 14 after challenge, and on day 14 kidneys, liver, and lungs were also sampled. Oral swabs were plated directly onto SDA containing 50 mg of chloramphenicol (Sigma) per ml (SDAC) for the selective qualitative recovery of C. albicans isolates. Fresh fecal samples (three fecal pellets per mouse) were collected, and kidneys, liver, and lungs were removed aseptically, weighed, homogenized in sterile saline, and serially diluted in saline and aliquots were spread onto SDAC. The plates were incubated for 48 h at 358C, and the results were recorded as the numbers of CFU per gram of tissue or feces. Oral swabs were scored subjectively

as 0 (no growth), 11 (1 to 49 CFU), 21 (50 to 99 CFU), 31 (100 to 199 CFU), or 41 (confluent growth; $200 CFU). Antifungal treatments. The antifungal compounds fluconazole (Pfizer, Groton, Conn.), nystatin (Sigma), and L-693989 and L-733560 (Merck Research Laboratories, Rahway, N.J.) were solubilized in 10 mM sodium citrate buffer (pH 5.0). Ketoconazole (Janssen, Beerse, Belgium) was solubilized in 5 ml of 0.2 N HCl and was then diluted in 10 mM sodium citrate (pH 5.0). The compounds were prepared fresh three times per week. Drugs were administered in the drinking water from days 3 to 13 postchallenge at concentrations of 400, 100, and 25 mg/ml. Because a 20-g mouse drinks approximately 5 ml of water per day (32), the dosages were approximately 100, 25, and 6.25 mg/kg/day. Sham-treated mice were anti-CD41 antibody- or hybridoma-treated mice administered gentamicin, challenged with C. albicans, and then administered 10 mM sodium citrate (pH 5.0) for the duration of antifungal treatment. In vitro antifungal susceptibility testing. The MICs of the compounds tested in the model described here for C. albicans MY1055 were determined by a colorimetric broth microdilution procedure modified from the National Committee for Clinical Laboratory Standards document M27-T (27). Briefly, by using the spectrophotometric method an inoculum of C. albicans MY1055 was made in RPMI 1640 medium buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (BioWhittaker, Walkersville, Md.) and containing 20% AlamarBlue (Alamar Biosciences, Inc., Sacramento, Calif.), an oxidation-reduction indicator. L-693989, L-733560, and fluconazole were solubilized in sterile water, and ketoconazole and nystatin were solubilized in dimethyl sulfoxide (Sigma). Microtiter plates containing serial dilutions of 100 ml of antifungal compounds in RPMI 1640 medium were inoculated with 100 ml of the adjusted inoculum. The final inoculum concentration was 0.5 3 103 to 2.5 3 103 CFU/ml, and the antifungal compound concentrations ranged from 128 to 0.06 mg/ml. The plates were incubated at 358C and were observed for the presence or absence of growth at 24 and 48 h. Growth was indicated by the change from dark blue to red, and the colorimetric MIC endpoints were determined to be the last well in which there was no color change. Statistics. The Student t test (two-tailed, unpaired, unequal variance) was used for the determination of significance. A P value of ,0.05 was considered significant.

RESULTS 1

CD4 T-lymphocyte reduction. Table 1 shows the CD41, CD81, and Thy-1.21 T-cell populations in GK1.5 hybridomatreated and anti-CD41 MAb-treated DBA/2 mice. Subcutaneous injection of 9 3 106 GK1.5 hybridoma cells significantly reduced the splenic CD41 T-cell population through 28 days compared with the population in untreated controls (P , 0.05). The CD41 T-cell population was completely depleted through day 21 (#0.2%), with the reappearance of CD41 cells at day 28 (7.5% 6 4.5%). The average splenic CD81 T-cell population in hybridoma-treated mice was not significantly changed compared with that in controls at any time point surveyed. The Thy-1.21 T-cell marker is expressed on 100% of the T lymphocytes in DBA/2 mice and includes both CD41 and CD81 T cells; therefore, a reduction in the CD41 population might result in a concomitant reduction in the Thy-1.21 population. A significant reduction in the Thy-1.21 T-cell population of hybridoma-treated mice occurred on days 14 (12.7% 6

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ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Broth microdilution MICs of antifungal compounds for C. albicans MY1055a MIC (mg/ml) Antifungal agent

Nystatin L-693989 L-733560 Fluconazole Ketoconazole

24 h

48 h

2 2 0.125 0.5 #0.06

8 2 0.25 0.5 #0.06

a The broth microdilution method was performed as described by National Committee for Clinical Laboratory Standards document M27-T (27) with RPMI 1640 medium buffered to pH 7.0 with 0.165 M MOPS and supplemented with 10% AlamarBlue.

FIG. 1. Effect of CD41 T-cell depletion on fecal recovery of C. albicans for gentamicin-treated DBA/2 mice.

1.5%) and 21 (13.5% 6 2.4%) (P , 0.05), but not on days 7 (19.9% 6 4.8%) and 28 (18.6% 6 2.6%). Three i.p. injections of purified anti-CD41 MAb, 300 mg on day 0, 200 mg on day 4, and 100 mg on day 7, significantly reduced the splenic CD41 T-lymphocyte population at all time points surveyed to 28 days (P , 0.05). CD41 T cells were completely depleted on day 7 (#0.1%), but began to reappear in the spleens of some MAb-treated mice at 14 days (1.1% 6 1.0%). The CD81 T-cell population was significantly increased in antibody-treated mice on day 7 only (P , 0.05), while the Thy-1.21 T-cell population was significantly decreased at all time points (P , 0.05). Both anti-CD41 treatments gave significant reductions in the CD41 T-cell population, but CD41 cells began to reappear 14 days earlier in antibody-treated mice than in hybridoma-treated mice. Only the CD41 T-cell population of antibody-treated mice at day 21 was significantly greater than that in hybridoma-treated mice. The remaining T-cell populations of the hybridoma- and antibody-treated groups were not significantly different at any time point. Since the CD41 T-cell population began to reappear at 14 days in antibody-treated mice, the amount of antibody was increased to 300 mg for all three injections in colonization studies, to be certain that the CD41 T-cell population remained depleted throughout the study. The CD41 T-cell depletion in colonized mice was confirmed by FACS analysis upon termination of the study at day 14 after challenge (average proportion of CD4 cells, 0.48%; data not shown). Alimentary tract colonization by C. albicans. (i) Fecal C. albicans isolates. C. albicans isolates were not detected in the feces or alimentary tract tissues (oral cavity, esophagus, stomach, small intestine, cecum, or large intestine) of a representative population of unchallenged, CD41 T-cell-deficient, gentamicin-treated DBA/2 mice (data not shown). In DBA/2 mice challenged orally with C. albicans, depletion of CD41 T cells by injection of anti-CD41 MAb in conjunction with gentamicin administration significantly increased the fecal recovery of C. albicans relative to that in mice treated with gentamicin only at all time points (P , 0.05) (Fig. 1). Despite increased and prolonged gastrointestinal colonization by C. albicans isolates, no dissemination of C. albicans organisms to other tissues (liver, lung, or kidney) was found. C. albicans organisms were detected in the feces of sham-treated mice at levels greater

than or equal to 4 3 104 CFU/g of feces through day 14 postchallenge. The MICs of the compounds tested in the model for C. albicans MY1055 are given in Table 2. The range of MICs at 48 h was from #0.06 to 8 mg/ml. The relative in vitro activity was ketoconazole . L-733560 . fluconazole . L-693989 . nystatin. Figure 2 shows the mean log CFU of C. albicans recovered per gram of feces from mice receiving antifungal treatment at levels of 400 mg/ml (Fig. 2A), 100 mg/ml (Fig. 2B), and 25 mg/ml (Fig. 2C) in the drinking water. At 2 and 7 days after the initiation of therapy at 400 mg/ml, the mean log CFU of C. albicans per gram of feces was reduced in mice treated with nystatin (3.73 on day 2 and 4.15 on day 7), L-693989 (2.85 on day 2 and 2.53 on day 7), ketoconazole (3.58 on day 2 and 3.02 on day 7), and fluconazole (3.55 on day 2 and 2.55 on day 7) compared with that in sham-treated controls (4.96 on day 2 and 4.70 on day 7). One day after the termination of therapy (day 11; data not shown) the log CFU of C. albicans per gram of feces in groups treated with fluconazole (2.96) and ketoconazole (2.95) remained reduced compared with that in shamtreated controls (4.61), while nystatin- and L-693989-treated mice no longer had reduced levels of fecal C. albicans organisms. With antifungal treatment at 100 mg/ml, nystatin and L-733560 reduced fecal colonization after 2 (3.97 and 3.17 log CFU/g, respectively) and 7 (3.67 and 2.50 log CFU/g, respectively) days of therapy, while fluconazole, ketoconazole, and L-693989 reduced fecal colonization significantly only at 7 days (3.08, 2.63, and 3.28 log CFU/g, respectively) relative to that in sham-treated controls (4.96 on day 2 and 4.70 on day 7). The mean log CFU of C. albicans per gram of feces in only fluconazole- and ketoconazole-treated mice remained reduced at 24 h after the termination of therapy (2.69 and 3.04, respectively). With antifungal treatment at 25 mg/ml in the drinking water, ketoconazole-treated mice had reduced levels of fecal C. albicans relative to those in sham-treated mice at all time points surveyed (3.75, 3.44, and 3.22 log CFU/g on days 2, 7, and 11, respectively). Fluconazole- and L-733560-treated mice had reduced levels of fecal C. albicans colonization compared with controls after 7 days of therapy (2.83 and 2.95 log CFU/g, respectively), and the log CFU of C. albicans per gram of feces in fluconazole-treated mice remained reduced compared with those in sham-treated controls at day 11, 1 day after the termination of therapy (2.95 log CFU/g). Fecal colonization in nystatin- and L-693989-treated mice was not significantly reduced compared with that sham-treated mice. (ii) Oral C. albicans isolates. Figure 3 shows the results of the semiquantitative recovery of C. albicans organisms from

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the oral cavities of mice treated with 400 mg (Fig. 3A), 100 mg (Fig. 3B), and 25 mg (Fig. 3C) of the antifungal compounds per ml in the drinking water. Reductions in the level of oral colonization were noted with all compounds at some point during therapy; however, only L-693989, fluconazole, and ketoconazole were able to eliminate the C. albicans organisms from the oral cavities of the mice. L-733560, fluconazole, and ketoconazole also reduced the level of oral colonization at earlier time points than nystatin and L-693989. A dose-response was seen, with decreasing concentrations of compounds taking longer to reduce oral colonization compared with that in sham-treated controls. DISCUSSION

FIG. 2. Effects of oral antifungal agents at 400 (A), 100 (B), and 25 (C) mg/ml on fecal recovery of C. albicans organisms from CD41 T-cell-deficient DBA/2 mice. p, significant reduction from the levels in sham-treated mice; Rx, treatment; NYS, nystatin; KTZ, ketoconazole; FCZ, fluconazole. Day 2 was the day after the initiation of treatment.

The increased incidence of mucosal candidiasis has been linked to a significant rise in the numbers of immune compromised patients (5, 15, 16, 21, 24, 25). Particularly, the decrease in CD41 T-lymphocyte counts associated with human immunodeficiency virus infection and AIDS has been correlated with the rise in cases of alimentary tract candidiasis (13, 15, 21, 30). Murine models of gastrointestinal candidiasis have also demonstrated the important role of CD41 T cells in resistance to colonization (2, 3, 6–8, 26). As the prevalence of mucocutaneous candidiasis and the incidence of azole-resistant strains of C. albicans increase, so will the need for novel antifungal agents. For the evaluation of these agents, we have developed a murine model of oropharyngeal and gastrointestinal colonization by C. albicans in mice specifically depleted of CD41 T lymphocytes to mimic the immune state of AIDS patients. Selective depletion of CD41 T lymphocytes in mice achieved either by the in vivo secretion of anti-CD41 MAb by GK1.5 hybridoma cells or by the direct injection of the purified antiCD41 MAb allowed colonization of the gastrointestinal tract by C. albicans isolates. Using FACS analysis, we have shown that one subcutaneous injection of 9 3 106 anti-CD41 MAbsecreting GK1.5 hybridoma cells is as effective at depleting CD41 T cells as three i.p. injections of 300 mg of purified anti-CD41 antibody. Although there were no differences in gastrointestinal colonization by C. albicans in mice depleted of CD41 T cells by either method, there were some side effects associated with each method. In long-term experiments, multiple injections of rat GK1.5 MAb may cause some mortality in mice (36). Although one injection of hybridoma cells requires less animal handling and is less labor intensive than MAb purification, it also has some disadvantages. Approximately 50% of DBA/2 mice injected with hybridoma cells developed visible tumors beginning 3 weeks following hybridoma injection (unpublished data). Many of these tumors became so large that they were lethal, while approximately one in eight tumors regressed. All mice with visible tumors were depleted of CD41 T cells, and depletion continued for up to 5 weeks after tumor regression. In mice with no visible tumors, CD41 T cells began to reappear at 4 weeks after injection of hybridoma cells. For experimental procedures requiring less than 3 weeks for their completion, these tumors pose no problem. However, in longterm experiments, significant mortality and the reappearance of CD41 T cells after 4 weeks must be anticipated. Although these tumors form in DBA/2 mice, in our experience with other strains of mice such as BALB/c and C3Heb/FeJ mice, visible tumors have not formed and CD41 T-cell depletion may have been more prolonged (22). After depletion of CD41 T lymphocytes and a short treatment with oral gentamicin, the alimentary tracts of the mice were colonized with C. albicans organisms. The severity of colonization and the efficacies of antifungal therapies were

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FIG. 3. Effects of oral antifungal agents at 400 (A), 100 (B), and 25 (C) mg/ml on oral recovery of C. albicans organisms from CD41 T-cell-deficient DBA/2 mice. Day 7 was the day after treatment (Rx) initiation. NYS, nystatin; KTZ, ketoconazole; FCZ, fluconazole. 0, 0 CFU; 1, 1 to 49 CFU; 2, 50 to 99 CFU; 3, 100 to 199 CFU; 4, $200 CFU.

ANTIMICROB. AGENTS CHEMOTHER.

easily monitored by culturing fecal pellets and oral swabs. Using this model, we have evaluated the efficacies of three representative classes of antifungal agents: polyenes, azoles, and echinocandins. The polyene nystatin remained a suspension in sodium citrate buffer and was relatively inactive in this model. Both azole antifungal agents tested, fluconazole and ketoconazole, as well as the water-soluble lipopeptides L-693989 and L-733560, rapidly reduced the fecal and oral Candida loads. The differences in the efficacies of the various compounds in this model correspond with the differences seen in the MICs of these compounds for C. albicans MY1055. The classic fungicidal and fungistatic modes of action of these compounds were not apparent in the model. For example, fluconazole, an agent known to be fungistatic, reduced the numbers of C. albicans CFU per gram of feces below the levels of detection (,49 CFU per sample) in 80% of the mice treated with the highest level of drug (400 mg/ml). Conversely, the polyene nystatin and the lipopeptides L-693989 and L-733560, known to have fungicidal mechanisms, were unable to reduce the numbers of C. albicans CFU below the levels of detection in more than 30% of the treated mice. The reason for this observation is unclear; however, the optimum formulations of these compounds were not addressed in the present studies and may have important ramifications for the delivery and maintenance of therapy. The CD41 T-cell-deficient mouse model described here was designed to mimic chronic colonization of the alimentary tract rather than tissue infection. Histological examination of specimens from the alimentary tracts of Candida-colonized mice showed no evidence of penetration of the epithelial cells by the yeast or hyphae nor evidence of inflammation. Candida colonization with small, self-limiting foci of mucosal involvement in the stomach was seen in 85% of DBA/2 mice intragastrically inoculated and administered antibiotics over a long term, but the C. albicans organisms eventually cleared without dissemination (4). Cantorna and Balish (6) have shown that a combination of both cell-mediated immunity and phagocytic cell defects is necessary for extensive infection or invasion of the gastrointestinal tract and subsequent dissemination of C. albicans (6). Gnotobiotic mice immunodeficient in either T-cell function (nu/nu) or phagocytic cell function (bg/bg) showed only low levels of infection with C. albicans in the gastrointestinal tract and no dissemination of C. albicans to other tissues, while doubly immunodeficient (bg/bg nu/nu) mice showed extensive infection of the gastrointestinal tract and the subsequent dissemination of C. albicans (6). This model of chronic colonization of the oropharynx and gastrointestinal tract by C. albicans in mice rendered CD41 T-lymphocyte deficient by GK1.5 hybridoma or purified anti-CD41 MAb injection mimics the immune state observed in AIDS patients and provides a simple method of assessing the therapeutic efficacies of antifungal compounds. In the present model, the echinocandins L-693989 and L-733560, as well as the azoles fluconazole and ketoconazole, showed superior efficacies relative to that of the polyene nystatin in reducing both oral and fecal Candida loads. Given the increase in the number of cases of esophageal candidiasis resistant to fluconazole therapy, especially in patients in advanced stages of AIDS, the novel echinocandin class of antifungal compounds may prove to be useful clinically for the treatment of oropharyngeal and gastrointestinal candidiasis. REFERENCES 1. Arendorf, T. M., and D. M. Walker. 1980. The prevalence and intra-oral distribution of Candida albicans in man. Arch. Oral Biol. 25:1–10. 2. Balish, E., M. J. Balish, C. A. Salkowski, K. W. Lee, and K. F. Bartizal. 1984. Colonization of congenitally athymic, gnotobiotic mice by Candida albicans. Appl. Environ. Microbiol. 47:647–652.

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