Validation of the Candida albicans delayed-type hypersensitivity (DTH) model in the female B₆C₃F₁ mouse for use in immunotoxicological investigations

Abstract

Although numerous models are used to evaluate the immunotoxic effects of xenobiotics on cell-mediated immunity (CMI), no holistic model for evaluating such effects on the delayed-type hypersensitivity (DTH) response has gained widespread acceptance. Due to a lack of interference from antigen-specific antibody production, the Candida albicans DTH model has recently been demonstrated to be a more appropriate model for assessing effects on CMI than other DTH models that utilize different sensitizing antigens, such as sheep erythrocytes (SRBC) or keyhole limpet hemocyanin (KLH). The present studies were conducted to validate the C. albicans DTH model for its ability to detect suppression (or the lack thereof) of CMI following exposure for 28 days to well-characterized immunosuppressive drugs, each having a different mechanism of action. The compounds evaluated included azathioprine (AZA), cyclophosphamide (CPS), cyclosporin A (CSA), dexamethasone (DEX), and the non-immunotoxic compound, benzo[e]pyrene (B[e]P). Exposure to each of the four known immunotoxicants resulted in statistically significant decreases in the DTH response to C. albicans. Footpad swelling was decreased following exposure to AZA at ≥ 20 mg/kg but not at 10 mg/kg, CPS at ≥ 10 mg/kg but not at 5 mg/kg, CSA at ≥ 3 mg/kg but not at 1 mg/kg, or DEX at ≥ 0.3 mg/kg (intermittently at 0.1 mg/kg) but not at 0.03 mg/kg. As expected, exposure to B[e]P for 28 days at doses up to 40 mg/kg had no effect on the DTH response. These results demonstrated that the C. albicans DTH assay in the B₆C₃F₁ mouse was capable of appropriately classifying each test article as to its immunotoxic effects on CMI. Furthermore, comparisons of these results with previous reports of effects on ex vivo CMI end points suggest that this DTH assay may be more sensitive than standard ex vivo assays at detecting immunosuppressive effects.

Keywords::

• Cell-mediated immunity
• delayed-type hypersensitivity (DTH) models
• immunotoxicity
• Candida albicans
• azathioprine
• cyclophosphamide
• cyclosporin A
• dexamethasone

Abbreviations
Ab,	=	Antibody
ANOVA,	=	Analysis of variance
AZA,	=	Azathioprine
B[a]P,	=	Benzo[a]pyrene
B[e]P,	=	Benzo[e]pyrene
BCG,	=	Bacillus Calmett-Guerin
BSA,	=	Bovine serum albumin
C. albicans,	=	Candida albicans
CHT,	=	Chitosan
CMI,	=	Cell-mediated immunity
CO,	=	Challenge only
Con A,	=	Concanavalin A
CPS,	=	Cyclophosphamide
CSA,	=	Cyclosporin A
CTL,	=	Cytotoxic T-lymphocyte
DEX,	=	Dexamethasone
DTH,	=	Delayed-type hypersensitivity
IP,	=	Intraperitoneal
KLH,	=	Keyhole limpet hemocyanin
MLR,	=	Mixed-leukocyte response
OVA,	=	Ovalbumin
PBS,	=	Phosphate-buffered saline
PHA,	=	Phytohemagglutinin
PO,	=	Per os
SC,	=	Subcutaneous
SE,	=	Standard error
SRBC,	=	Sheep red blood cells
VH,	=	Vehicle

Introduction

In current immunotoxicology testing protocols, holistic evaluations of the effects of a drug or chemical on humoral immunity are typically conducted utilizing either of the T-dependent antigens, sheep erythrocytes (SRBC) or keyhole limpet hemocyanin (KLH), in the plaque assay for SRBC (Jerne et al., 1963; White et al., 2010) or in an enzyme-linked immunosorbent assay (ELISA) for either SRBC or KLH (Temple et al., 1993; Plitnick and Herzyk, 2010). However, there is no standardized, holistic assay for examining immunotoxic effects on cell-mediated immunity (CMI). Instead, CMI is usually evaluated in ex vivo lymphoproliferative assays using T-cell mitogens, anti-CD3 antibody-mediated stimulation, and the mixed-leukocyte response (MLR). In addition, the ex vivo cytotoxic T-lymphocyte (CTL) assay is also used to evaluate the cytolytic capabilities of effector CD8⁺ T-cells.

The delayed-type hypersensitivity (DTH) response has been well characterized as a cell-mediated response (Black, 1999; Kaufmann and Schaible, 2005). However, given the number of different DTH models being utilized, there appears to be little agreement among immunotoxicologists when it comes to the choice of sensitizing and challenge antigens to be used. Among the antigens utilized in the DTH, from the published literature, are: tetanus toxoid, Candida albicans, Mycobacterium bovis [Bacillus Calmett-Guerin (BCG)], SRBC, bovine serum albumin (BSA), KLH, and ovalbumin (OVA) (Lagrange and Mackaness, 1975; Vos, 1980; Whittingham et al., 1982; Henningsen et al., 1984; Hurtrel et al., 1984; Exon et al., 1990; Nghiem et al., 2002). Unfortunately, many of these antigens, such as SRBC, BSA, OVA, and KLH, are also capable of initiating a robust humoral immune response, and the negating effects of antigen-specific antibody (Ab) production on the DTH response are well-documented (Mackaness et al., 1974; Morikawa et al., 1991). The ideal antigen for evaluating the DTH response would therefore be one that produced a robust cell-mediated response but did not produce an antigen-specific antibody. This would allow for the evaluation of the effects of a xenobiotic on CMI without interference from confounding factors resulting from antibody production that potentially could obscure the immunomodulating effects of the test compound.

Smith and White (2010) have recently shown that the use of formalin-fixed C. albicans as the sensitizing antigen followed by challenge with chitosan produced a cell-mediated response with no detectable antigen-specific Ab component. The challenge agent, chitosan, is a commercially available purified extract from the cell wall of C. albicans that produces minimal non-specific swelling following injection into the footpad and does not elicit a humoral immune response.

The objective of this research was to validate the C. albicans DTH model in the B₆C₃F₁ mouse for its ability to detect suppression (or the lack thereof) of CMI following exposure for 28 days to one of four well-characterized immunosuppressive drugs, each having a different mechanism of action. The compounds evaluated were: azathioprine (AZA), cyclophosphamide (CPS), cyclosporin A (CSA), dexamethasone (DEX), and the non-immunosuppressive polycyclic aromatic hydrocarbon, benzo[e]pyrene (B[e]P). The hypothesis was that each of the four immunosuppressive drugs would produce suppression of the footpad swelling response following 28 days of exposure, while the non-immunotoxic compound (B[e]P) would not affect the DTH response. Due to the complex nature of the DTH response in vivo, we also hypothesized that the DTH assay should have equal or greater sensitivity than the currently used ex vivo CMI assays. Utilizing the published work of Lebrec et al. (1994), we were able to compare the sensitivity of the holistic C. albicans DTH model in the B₆C₃F₁ mouse to several of the ex vivo CMI assays.

Materials and methods

Animal husbandry

Female B₆C₃F₁ mice (8-12 weeks of age) from Taconic Farms (Germantown, NY) were used in these studies. Animals were quarantined for 1 week prior to use and were determined to be free of hepatitis and Sendai virus by serology testing. Mice were given access to NTP 2000 Laboratory Diet and tap water ad libitum. Ambient temperatures were maintained at 21–24°C, relative humidity was maintained between 40–70%, and a 12-h light/dark cycle was utilized. Mice were randomly assigned to respective treatment groups (eight animals per group). All animal procedures were conducted in an AAALAC-accredited facility under an animal protocol approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee (IACUC).

Test articles and animal exposure

All animal exposures to the test article were administered in a volume of 0.1 ml/10 g body weight. AZA (Sigma Aldrich, St. Louis, MO) was administered by oral gavage (PO) in 0.5% methylcellulose vehicle (VH) at doses ranging from 3–30 mg/kg. B[e]P (Sigma) was given by subcutaneous injection (SC) at 5–40 mg/kg, using corn oil as the VH. CPS (Sigma) was prepared in phosphate-buffered saline (PBS, pH 7.4) VH and administered at 3–30 mg/kg by intraperitoneal (IP) injection. CSA (Novartis Pharmaceuticals Corp., East Hanover, NJ) was administered PO at 0.3–30 mg/kg in corn oil VH. DEX (Sigma) was administered PO as a suspension in the VH 0.5% methylcellulose with 0.2% Tween 80 at doses ranging from 0.03–1.00 mg/kg.

Evaluation of the delayed-type hypersensitivity (DTH) response to C. albicans

The DTH response to C. albicans was evaluated as described by Smith and White (2010). Briefly, formalin-fixed C. albicans (AlerChek Inc., Portland, ME) were diluted to 1 × 10⁸ organisms/ml in 0.9% NaCl and administered in a volume of 0.1 ml/mouse SC into the right flank on Day 21 of the study. On Day 29, 1 day after the final exposure to the test article, pre-measurements of the right footpad were made with a digital micrometer (Mitutoyo Corp., Tokyo, Japan), and mice were subsequently challenged with the C. albicans antigen, chitosan (CHT; AlerChek Inc.), by SC injection into the right footpad in a volume of 0.04 ml (1 mg/ml). Twenty-four (± 2) hours post-challenge, the thickness of the right footpad was measured, and the footpad swelling for each mouse was calculated (average post-challenge thickness−average pre-challenge thickness). Data are reported in terms of footpad swelling in units of mm × 100. Also included in each study was a group of mice that were challenged with CHT in the footpad on Day 29 but were neither exposed to the test article nor sensitized with C. albicans. This “challenge only” group (CO) was used to determine the background footpad swelling resulting from the CHT challenge and also to demonstrate that the footpad swelling observed in the VH control group was the result of an immunological memory response (as opposed to an innate, inflammatory response). CPS was used as a positive control in studies where no effect was anticipated in order to insure the assay system was functioning appropriately. In such experiments, CPS was administered on Days 25–28 at 50 mg/kg by IP injection.

Statistical analysis

Results represent the mean ± standard error (SE) obtained from combining multiple studies, each of which utilized seven or eight animals/group. Statistical analysis of all data was performed using JMP^™ 5.0 (SAS Institute, Inc., Cary, NC) by first using Bartlett’s test for homogeneity of variances, followed by an analysis of variance (ANOVA) (parametric or non-parametric as necessary). Ad hoc pair-wise comparisons were made using Dunnett’s test for parametric data. Pair-wise comparisons between exposure groups and vehicle control for non-parametric data were made using the Wilcoxon Rank Test. Comparisons of vehicle control means between multiple studies evaluating the same compound were conducted using the Student’s t-test. In all evaluations, p < 0.05 indicated statistically significant differences.

Results

Effects of AZA exposure

Mice were exposed to AZA for 28 days at doses of 3, 5, 10, 20, or 30 mg/kg. Statistically-significant decreases in the DTH response to C. albicans were observed at 20 mg/kg AZA and greater (Figure 1). Footpad swelling was significantly decreased by 49% for the 20 mg/kg AZA group and by 68% at the high dose (30 mg/kg) as compared to the VH control. No effects were observed on the DTH at lower doses of AZA (i.e. ≤ 10 mg/kg). No effects on terminal body weights were observed at doses up to and including 20 mg/kg AZA (data not shown). AZA exposure at the high dose (30 mg/kg) was overtly toxic, as evidenced by a jaundiced discoloring of the skin and by significant decreases (9%) in terminal body weight.

Figure 1.  Effects of AZA exposure on the DTH response to C. albicans. Mice were administered either vehicle (0.5% methylcellulose) or AZA PO daily for 28 days. Mice were sensitized on Day 21 with C. albicans. The right footpad of each mouse was pre-measured and challenged with chitosan antigen on Day 29, with post-measurement occurring 24 [± 2] h after challenge. The background footpad swelling was determined in a group of mice that were challenged but not sensitized (challenge only). The data are expressed as footpad swelling (mm × 100). Values represent the mean (± SE) of two combined studies, each containing 7–8 animals per group. Vehicle control means between studies were not statistically significantly different (57.1 [± 10.0] vs 53.4 [± 11.7] mm × 100). ** p < 0.01.

Effects of CPS exposure

Mice were exposed to CPS at doses of 3, 5, 10, 20, or 30 mg/kg for 28 days. A dose-related suppression of the DTH response to C. albicans was observed at 10 mg/kg and greater dose levels, while no effects were observed at 5 mg/kg and lower (Figure 2). Footpad swelling was decreased by 28% for the 10 mg/kg CPS group, by 61% for the 20 mg/kg CPS group, and by 86% for the 30 mg/kg CPS group, as compared to VH control. CPS exposure did not result in effects on terminal body weight at any dose (data not shown).

Figure 2.  Effects of CPS exposure on the DTH response to C. albicans. Mice were administered either vehicle (PBS) or CPS IP daily for 28 days. Mice were sensitized on Day 21 with C. albicans. The right footpad of each mouse was pre-measured and challenged with chitosan antigen on Day 29, with post-measurement occurring 24 [± 2] h after challenge. The background footpad swelling was determined in a group of mice that were challenged but not sensitized (challenge only). The data are expressed as footpad swelling (mm × 100). Values represent the mean (± SE) of two combined studies, each containing 7–8 animals per group. Vehicle control means between studies were not statistically significantly different (78.4 [± 9.2] vs 62.9 [± 7.1] mm × 100). * p < 0.05; ** p < 0.01.

Effects of CSA exposure

CSA exposure was conducted daily for 28 days at dose levels of 0.3, 1, 3, 10, or 30 mg/kg. Exposure produced statistically significant immunosuppressive effects on the DTH response at 3 mg/kg CSA and higher, while no effects were observed at lower doses (0.3–1.0 mg/kg; Figure 3). When compared to the VH control group, footpad swelling was decreased by 33% at 3 mg/kg CSA, by 34% at 10 mg/kg CSA, and by 36% at 30 mg/kg CSA. No effects were observed on terminal body weights at any dose level of CSA (data not shown).

Figure 3.  Effects of CSA exposure on the DTH response to C. albicans. Mice were administered either vehicle (corn oil) or CSA PO daily for 28 days. Mice were sensitized on Day 21 with C. albicans. The right footpad of each mouse was pre-measured and challenged with chitosan antigen on Day 29, with post-measurement occurring 24 [± 2] h after challenge. The background footpad swelling was determined in a group of mice that were challenged but not sensitized (challenge only). The data are expressed as footpad swelling (mm × 100). Values represent the mean (± SE) of two combined studies, each containing 7–8 animals per group. Vehicle control means between studies were not statistically significantly different (78.4 [± 8.0] vs 65.1 [± 10.4] mm × 100). ** p < 0.01.

Effects of DEX exposure

Daily exposure to DEX was conducted for 28 days at 0.03, 0.1, 0.3, 0.5, or 1 mg/kg. Significant decreases in footpad swelling were observed at dose levels of 0.3, 0.5, and 1 mg/kg (Figure 4). Swelling was decreased by 58% at 0.3 mg/kg, 56% at 0.5 mg/kg, and 70% at 1 mg/kg when compared to the VH control. No effects were observed at 0.03 mg/kg in any of the studies conducted. Intermittent suppression was observed at 0.1 mg/kg; however, the decrease at this dose level failed to reach the level of statistical significance (p = 0.07) when the studies were evaluated together. No effects were observed on terminal body weights at any DEX dose evaluated (data not shown).

Figure 4.  Effects of DEX exposure on the DTH response to C. albicans. Mice were administered either vehicle (0.5% methylcellulose) or DEX PO daily for 28 days. Mice were sensitized on Day 21 with C. albicans. The right footpad of each mouse was pre-measured and challenged with chitosan antigen on Day 29, with post-measurement occurring 24 [± 2] h after challenge. The background footpad swelling was determined in a group of mice that were challenged but not sensitized (challenge only). The data are expressed as footpad swelling (mm × 100). Values represent the mean (± SE) of two combined studies, each containing 7–8 animals per group. Vehicle control means between studies were not statistically significantly different (71.0 [± 7.6] vs 60.3 [± 7.9] mm × 100. ** p < 0.01.

Effects of B[e]P exposure

B[e]P exposure at doses of 5, 20, and 40 mg/kg for 28 days did not affect the DTH response to C. albicans (Figure 5). No significant effects were observed on terminal body weights following B[e]P exposure at any dose (data not shown).

Figure 5.  Lack of effect of B[e]P exposure on the DTH response to C. albicans. Mice were administered either vehicle (corn oil) or B[e]P SC daily for 28 days. Positive control mice were administered (IP) 50 mg/kg CPS on Days 25–28. Mice were sensitized on Day 21 with C. albicans. The right footpad of each mouse was pre-measured and challenged with chitosan antigen on Day 29, with post-measurement occurring 24 [± 2] h after challenge. The back-ground footpad swelling was determined in a group of mice that were challenged but not sensitized (challenge only). The data are expressed as footpad swelling (mm × 100). Values represent the mean (± SE) from eight animals per group.

Comparison of the sensitivity of the holistic C. albicans DTH model with ex vivo studies of Lebrec et al. (1994)

Although it would be desirable to do side-by-side comparisons of the lymphoproliferative assays and CTL assay with the DTH response to C. albicans in the same laboratory, comparing our DTH results with the published work of Lebrec et al. (1994) is consistent with the goals of reducing animal usage and avoiding the unnecessary repeating of studies that have already been conducted. Accordingly, we compared the sensitivity of our in vivo DTH findings in the B₆C₃F₁ mouse to the ex vivo studies of Lebrec et al. (1994), which were conducted in the same sex and strain of mouse and utilized several of the same compounds and similar dose levels as our present work. Table 1 summarizes the effect and no observable effect levels (NOEL) for both the C. albicans DTH studies and the Lebrec et al. studies. For each of the cell-mediated immunotoxicological end-points evaluated by Lebrec et al., the dose at which the C. albicans DTH detected immunosuppression was less than or equal to the dose level reported by Lebrec et al. for the ex vivo CMI assays.

Table l.  Comparison of the C. albicans DTH model with ex vivo studies by Lebrec et al. (1994).

	CPS (mg/kg/day)		DEX (mg/k g/day)		AZA (mg/kg/day)		CSA (mg/kg/day)
	Effect Level	NOEL	Effect Level	NOEL	Effect Level	NOEL	Effect Level	NOEL
C. albicans DTH	≥10	5	≥0.3	0.03	≥20	10	≥3	1
			(intermittant effects at 0.1)
Doses Utilized	3, 5, 10, 20, 30		0.03, 0.1, 0.3, 0.5, 1		3, 5, 10, 20, 30		0.3, 1, 3, 10, 30
Ex vivo Endpoints
Lebrec et al. (1994)
Con A	Not Conducted		0.5	0.1	NE	20	50	25
PHA	Not Conducted		0.5	0.1	NE	20	≥12.5	ND
Anti-CD3	Not Conducted		0.5	0.1	NE	20	50	25
MLR	Not Conducted		NE	0.5	NE	20	≥25	12.5
CTL	Not Conducted		0.5^a	0.1	NE	20	≥25	125
Doses Utilized	Not Conducted		0.02, 0.1, 0.5		0.8, 4, 20		12.5, 25, 50

^aStudy showed an effect in this parameter at a lower dose, but it was not observed at a higher dose. NE = NoEffect, ND = Not Determined.

Discussion

When evaluating the immunotoxic effects of a xenobiotic on CMI, one of the suggested approaches is to employ ex vivo assays, including lymphoproliferative assays (e.g. concanavalin A [Con A] or anti-CD3-mediated proliferation), the MLR, and the CTL (Luster et al., 1988). One of the problems associated with the ex vivo approach is that, once cells are removed from the animals, there is the possibility that the cells can recover from the effects of the drug or chemical, particularly if the assay takes several days in culture to complete. The DTH assay, unlike the above ex vivo assays, provides an in vivo, holistic evaluation of CMI, and thus recovery is not a concern. However, there is currently no widespread acceptance of any single DTH model, specifically with regard to the choice of the sensitizing and challenge antigens.

We have recently published regarding three of the more popular DTH models (KLH, SRBC, and C. albicans), where we demonstrated that the C. albicans model, in addition to being the most sensitive model to the immunosuppressive effects of benzo[a]pyrene (B[a]P), produced a cell-mediated response with no interference from humoral immunity (Smith and White, 2010). The present research was designed to further validate this model for its ability to detect toxicant-induced changes to CMI. In these studies, suppression of footpad swelling was observed for each of the four immunosuppressive drugs at doses that did not produce a general toxic effect (as evidenced by a lack of treatment-related effects on terminal body weight), thus indicating that this model is capable of detecting immune system-specific effects.

The compounds evaluated in the present studies are well-documented immunosuppressive drugs, each having a different mechanism of action. Of the four drugs evaluated, the alkylating agent, CPS, produced a dose-related response with the greatest degree of immunosuppression as compared to the other drugs. When CPS was administered at a dose of 30 mg/kg, the DTH response was suppressed by 86%. The 30 mg/kg CPS dose did not produce a statistically significant decrease in terminal body weight. Administration of the potent synthetic glucocorticoid, DEX, produced the next greatest degree of suppression, in a dose-related manner, where a dose of 1 mg/kg decreased the DTH response by 70%. This dose of DEX did not statistically alter terminal body weights. The anti-metabolite, AZA, produced the third greatest dose-related degree of immunosuppression, where a dose of 20 mg/kg resulted in a 49% suppression of the footpad swelling response. At the highest dose level (30 mg/kg), in addition to suppression of the C. albicans DTH response, statistically significant decreases in body weights were observed, thus, the penultimate dose (20 mg/kg) was used for comparison purposes.

CSA, which predominantly alters T-cell cytokine production through the inhibition of calcineurin following the complexing of CSA with cyclophylins, is a more selective immunosuppressant than the other three compounds. Administration of CSA produced a 36% decrease at a dose of 30 mg/kg, which did not statistically alter body weights. The plateauing of the footpad swelling response observed in the CSA studies contrasts with the shape of the dose–response curve of the three other less selective immunosuppressive drugs evaluated. This may be due to the fact that the DTH reaction consists of a number of infiltrating cell types, including neutrophils, macrophages, and T-cells (Black, 1999), each of which has been demonstrated to participate in the DTH response to C. albicans (Smith and White, 2010). Furthermore, CSA does not affect chemotaxis or phagocytosis by either neutrophils or macrophages (Borel et al., 1976; Kharazmi et al., 1985). This lack of effect on neutrophils and macrophages may explain the plateauing of the footpad swelling response observed in the present CSA studies, a phenomenon which has also been previously reported in oxazolone hypersensitivity studies following CSA treatment (Borel et al., 1977).

In the present studies, the immunosuppressive effects of AZA and CSA were detected in the C. albicans DTH model at doses lower than previously reported CMI no-effect levels (NOEL) obtained from ex vivo studies, while, for DEX, effects were observed at similar doses to previous reports (Lebrec et al., 1994). CPS was not evaluated in the Lebrec et al. studies. The C. albicans DTH model is a holistic, in vivo functional assay for evaluating CMI that appears to be at least as sensitive as (if not more sensitive than) the standard ex vivo assays at detecting suppression of lymphoproliferation. Furthermore, the DTH assay measures additional parameters than just proliferation. The DTH is an end-line assay requiring the uptake, processing, and presentation of antigen followed by the maturation of antigen-specific T-cells to effector cells, which, upon subsequent antigen exposure, are capable of proliferation and release of immunological mediators that result in the recruitment of multiple additional cell types to the site of challenge, vascular leakage, and subsequent footpad swelling.

The sensitivity of the C. albicans DTH model contrasts with older reports suggesting that the DTH is not a sensitive indicator of immunotoxicity (Vos, 1980; Luster et al., 1988). However, the standard DTH model of choice at that time was the KLH model, which has been shown to be less sensitive than the C. albicans DTH at detecting immunosuppression (Smith and White, 2010). Although Luster et al. (1988) classify the DTH as a Tier II assay and suggest that a DTH be conducted only if an effect is seen in Tier I endpoints (Con A and MLR), the present studies suggest that the use of the C. albicans DTH assay may allow for the detection of a xenobiotic effect on CMI at doses that may not impact ex vivo functional assays. One of the reasons for the placement of the DTH in the Tier II assay paradigm suggested by Luster et al. was the poor reproducibility of results between four laboratories using the KLH DTH assay when evaluating the same compound. Unlike today, where a purified preparation of KLH is commercially available, at the time the evaluations reported by Luster et al. (1988) were conducted, each laboratory had to purify and prepare the KLH from unrefined starting material such that each preparation was different.

Finally, the validation of the C. albicans DTH assay in the adult mouse, presented here, segues to the use of the C. albicans DTH assay for developmental immunotoxicology (DIT) studies. The establishment of a DTH model for use in DIT studies has been identified as an important data gap and has been recommend to receive the highest priority for new model development in several DIT workshops (Holsapple, 2002; Holsapple et al., 2005; Burns-Naas et al., 2008). The C. albicans DTH assay, following adequate validation, should meet this need.

Conclusions

The C. albicans DTH model correctly identified each compound evaluated herein as either immunotoxic (AZA, CPS, CSA, and DEX) or non-immunotoxic (B[e]P). Furthermore, the immunosuppressive drugs each produced significant decreases in the DTH response at doses equal to or lower than previously reported effect levels from ex vivo CMI assays, suggesting that this DTH model may be more sensitive than ex vivo assays at detecting suppression of CMI.

The authors wish to extend special thanks to Michael Montague for excellent technical assistance. Thanks also to Ronnetta Brown, Deborah Musgrove, Anthony Brown, Julia Nims, and Andre Savage.

Declaration of interest

Dr Kimber L. White, Jr. is the owner of a company, ImmunoTox^®, Inc., that conducts immunotoxicological studies under Good Laboratory Practices (GLP). This work was supported in part by the National Institute of Environmental Health Sciences [ES 55538].