Activity of Voriconazole Combined with Neutrophils or Monocytes against Aspergillus fumigatus: Effects of Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor

Abstract

Voriconazole (VCZ) was tested for antifungal activity against Aspergillus fumigatus hyphae alone or in combination with neutrophils or monocytes. Antifungal activity was measured as percent inhibition of hyphal growth in assays using the dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] or XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide]. With both assays, VCZ inhibited hyphal growth at concentrations of <1 μg/ml and was almost as active as amphotericin B. VCZ (0.6 μg/ml) was sporicidal, as was amphotericin B (0.4 μg/ml). With both the MTT and XTT assays, neutrophils alone inhibited hyphae; when combined with VCZ, there was additive activity. Both granulocyte colony-stimulating factor- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-treated polymorphonuclear neutrophils (PMN) had enhanced inhibition of hyphal growth. Moreover, such treatment of PMN also enhanced the collaboration of PMN with VCZ. Monocytes inhibited hyphal growth. When VCZ was combined with monocytes or monocytes were treated with GM-CSF, inhibition was significantly increased, to similar levels. However, the combination of VCZ with GM-CSF treatment of monocytes did not significantly increase the high-level inhibition by monocytes with either agent alone.

Aspergillosis continues to be a frequent and difficult-to-treat fungal infection in certain immunocompromised patients (4). Although most Aspergillus species are susceptible to amphotericin B (AmB) or itraconazole in vitro (3), development of new antifungal agents with less toxicity, better solubility, and desirable pharmacokinetics is important. Of potential interest is the development of a new oral wide-spectrum triazole, voriconazole (VCZ), which has in vitro activity against Aspergillus species (10).

Previously, we have reported synergy of phagocytic cells with fluconazole for enhanced killing of Candida albicans (1, 5, 6), which reflects in vivo efficacy. Here, we tested the possibility that phagocytic cells could collaborate with VCZ for enhanced antifungal activity against Aspergillus fumigatus. Moreover, we investigated the effects that granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment might have on antifungal activity of neutrophils or monocytes for A. fumigatus and possible collaboration with VCZ for enhanced antifungal activity.

MATERIALS AND METHODS

A. fumigatus.

Two clinical isolates (92-270 and 96-92) were used principally in these studies. Isolates were grown on agar slants at 35°C and then allowed to form conidia at room temperature for 24 to 48 h. Conidia were harvested in distilled water, washed, diluted in saline, and counted. Conidial suspensions consisted primarily of single conidia (95%); the remainder were clumps of two or three conidia. These were dispensed into 24-well tissue culture plates or 96-well microtest plates. Over 90% germinated when incubated overnight in RPMI 1640 at 26 to 37°C; hyphal lengths ranged up to 10 times the diameter of a conidium. For studies on hyphae, material from overnight growth at 30°C (germlings) was used.

Activity of drugs on conidia was assessed by incubation of conidia in RPMI 1640 at 10³/well in a microtest plate and subculturing of well contents on blood agar plates.

MTT and XTT.

Inhibition of hyphal growth was measured by the colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma, St. Louis, Mo.) assay (8). MTT that was metabolized to formazan by viable hyphae was extracted with acidified isopropanol, and absorbance (A) was measured at 570 nm (A₅₇₀) with a Shimadzu (Kyoto, Japan) UV 160 spectrophotometer. Percent inhibition of growth was calculated by the formula {[A (control) − A (experimental)]/A [control]} × 100.

Inhibition of hyphal growth was also measured by the colorimetric XTT-plus-coenzyme Q method (9). XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] sodium salt at 0.5 mg/ml, coenzyme Q (2,3-dimethoxy-5-methyl-1,4-benzoquinone) at 0.04 mg/ml, and phosphate-buffered saline (PBS) (pH 7.4) constituted the test solution. Viable cells reduce XTT to a reduced soluble form with a color change from yellow to orange. Absorbance of XTT solution alone at 410 nm was subtracted from absorbance of metabolized XTT in culture supernatants at 410 nm to give the change in absorbance (ΔA), evaluated by a Dynatech (Chantilly, Va.) MR250 microtest plate reader. Percent inhibition was calculated by the formula {[ΔA (control) − ΔA (experimental)]/ΔA [control]} × 100.

VCZ and AmB.

VCZ (Pfizer, Groton, Conn.) was dissolved in dimethyl sulfoxide and then diluted with distilled water to 2 mg/ml and stored at 4°C. Desired dilutions were made from the stock solution with RPMI 1640. AmB (Fungizone; Squibb and Sons Inc., Princeton, N.J.), kept refrigerated or frozen and protected from light in distilled water at 1.6 mg/ml, was diluted in RPMI 1640 to give appropriate concentrations for testing.

G-CSF and GM-CSF.

Recombinant methionyl human G-CSF (Filgrastim) was provided by Amgen, Thousands Oaks, Calif. G-CSF at 10⁸ U/mg of protein was supplied at 0.3 mg/ml, and appropriate dilutions from this stock were made in RPMI 1640. Recombinant human GM-CSF (Leukine, Sargramostim) was produced and supplied by Immunex Corp., Seattle, Wash. GM-CSF (0.5 mg/ml; 1.5 × 10⁸ IU/mg of protein) was diluted to 7.5 × 10⁵ IU/ml in RPMI 1640 and stored at −80°C.

Neutrophil assays.

Polymorphonuclear neutrophils (PMN) and peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by sedimentation in 6% dextran-70 followed by density gradient centrifugation on Histopaque 1077 (Sigma). For the MTT assay, PMN were suspended to 2 × 10⁶/ml of CTCM (RPMI 1640 plus 10% fresh human serum), and 1 ml was added to wells containing germinated conidia (10⁶/well), giving an approximate effector-to-target (E/T) ratio of 10:1. Some sets of quadruplicate cultures received 0.01 ml of a G-CSF dilution. Other sets of quadruplicate cultures without PMN, with PMN, or with PMN and G-CSF received 0.01 ml of AmB or VCZ to the desired final concentrations of drugs.

After 24 h of incubation at 37°C in a CO₂ incubator, cultures were harvested into 15-ml conical centrifuge tubes with distilled water to lyse PMN. Washed hyphae from each well were incubated in 1 ml of RPMI plus MTT (0.5 mg/ml) for 3 h at 37°C. Following incubation, hyphae were pelleted by centrifugation, supernatants were aspirated, and metabolized MTT (formazan) in each tube was extracted with acidified isopropanol for 5 h at 37°C. Solubilized formazan from each tube was transferred to individual wells of a 96-well flat-bottom microtest plate, and absorbance was measured at 570 nm.

For the XTT assay, germinated conidia (1 × 10³ to 5 × 10³/well) in 96-well microtest plates were centrifuged in situ (400 × g; 10 min), supernatants were aspirated, and 0.2 ml of PMN at 2.5 × 10⁵ to 2 × 10⁶ per ml of CTCM was added per coculture well, giving 5 × 10⁴ to 4 × 10⁵ per well, respectively. Some cocultures received VCZ, G-CSF, or GM-CSF alone, while other sets of cocultures received G-CSF plus VCZ or GM-CSF plus VCZ. Following incubation at 37°C for 24 h in a CO₂ incubator, microtest plates were centrifuged, supernatants were aspirated, and 0.2 ml of distilled water per well was added. The centrifugation and washing step was repeated once more. Finally, 0.2 ml of XTT test solution per well was added, and cultures were incubated for 1 h at 37°C. The microtest plate was centrifuged again, 0.1 ml of supernatant from each well was transferred to a well of a new plate, and absorbance at 410 nm was recorded.

Monocyte assay.

PBMC at 5 × 10⁶/ml of CTCM were dispensed at 1 ml per well of 24-well tissue culture plates and then incubated for 2 h at 37°C in a CO₂ incubator. After incubation, nonadherent cells were aspirated and wells were washed once with RPMI 1640. Monocyte monolayers in 1 ml of CTCM were challenged with 1 ml of germinated conidia (10⁴/ml). To some sets of duplicate cultures, 0.01 ml of GM-CSF was added to give the desired final concentrations. Other sets of duplicate cultures without monocytes, with monocytes, or with monocytes plus GM-CSF received 0.01 ml of VCZ to give the desired final concentrations. After cultures were incubated for 24 h at 37°C in a CO₂ incubator, they were harvested and processed as described above for the PMN MTT assay.

Statistical analysis.

Student’s t test was used for statistical analysis of data, and significance was set at P of <0.05. The GB-STAT program (Microsoft, Redmond, Wash.) for Bonferroni’s adjustment to the t test was used where appropriate.

RESULTS

Preliminary tests.

Initial experiments determined the parameters required for optimal results in the MTT assay. Germination and incubation of increasing numbers of conidia in wells of 24-well tissue culture plates showed that absorbance of the solubilized metabolic product of MTT metabolism increased linearly with the number of conidia (Fig. 1). These results suggested that reliable results would be obtained by testing the antifungal activities of drugs or phagocytic cells in wells of tissue culture plates.

FIG. 1.

Inoculum size and MTT metabolism by A. fumigatus (isolate 92-270) in 12-well tissue culture plates. The numbers of conidia per well at time zero are given on the x axis. Conidia in RPMI 1640 were incubated for 24 h at 37°C. The y axis shows absorbance by 1 ml of metabolized MTT (means and standard deviations of four samples) measured at 570 nm with a spectrophotometer.

Initial experiments with the XTT assay were done with a range of conidium inocula, which were cultured in wells of a 96-well microtest plate at 37°C for 24 h in RPMI 1640. In the XTT assay, there was a linear relationship between the metabolism of XTT and the inoculum size, as shown in Fig. 2. This in situ method was less complicated and more sensitive than the MTT method.

FIG. 2.

Inoculum size and XTT metabolism by A. fumigatus (isolate 96-92) in microtest plate wells. The numbers of conidia per well at time zero are given on the x axis. Conidia in RPMI 1640 were incubated for 24 h at 37°C. The y axis shows absorbance by 0.1 ml of metabolized XTT (1 h at 37°C) by hyphae (means and standard deviations of four samples) measured at 410 nm with a microtest plate reader.

Activity of VCZ for conidia.

VCZ at 0.6 to 5.0 μg/ml and AmB (0.4 to 5.0 μg/ml) in RPMI 1640 at 26 or 37°C for 24 or 48 h were each fungicidal for conidia, i.e., there was no growth in subculture. Microscopic examination showed that the conidia did not germinate at these concentrations; however, lower, nonfungicidal concentrations of these drugs (VCZ, ≤0.4 μg/ml; AmB, ≤0.2 μg/ml) did not prevent germination.

Activity of VCZ with or without PMN against germlings: MTT assay.

VCZ at 0.5 μg/ml in the MTT assay caused significant inhibition (41%) of hyphal growth in 24 h (Table 1). In a total of four experiments with 1 × 10⁵ to 2 × 10⁵ germlings/well, VCZ at 0.5 μg/ml inhibited growth by 50% ± 7%. PMN alone inhibited hyphal growth of A. fumigatus by 30%. When VCZ and PMN were combined, inhibition (75%) was additive (Table 1). By contrast, the combination of AmB and PMN did not increase inhibition over that of AmB alone. Whether this was due to the highly effective activity of AmB alone or to some other effect remains to be determined.

TABLE 1.

Collaboration of VCZ or AmB with PMN for A. fumigatus hyphal growth inhibition: MTT assay

Condition	A570 (n = 4)a	% Inhi- bition	Pb
			Ic	IId	IIIe
Medium (control)	0.539 ± 0.128	0	R
VCZ (0.5 μg/ml)	0.319 ± 0.105	41	<0.05
PMN (2 × 105/well)	0.381 ± 0.114	30	NS	R
PMN + VCZ	0.137 ± 0.010	75	<0.01	<0.01f
AmB (0.5 μg/ml)	0.142 ± 0.030	74	<0.01		R
PMN + AmB	0.228 ± 0.050	58	<0.01	<0.01	NS

^aMTT metabolism by hyphae (isolate 92-270) after exposure to VCZ, AmB, PMN plus VCZ, or PMN plus AmB for 24 h at 37°C (10⁵ germlings/well). Values are means ± standard deviations. 

^bR, reference value; NS, not significant (P > 0.05). 

^cMetabolism of MTT in medium (control) cultures compared to MTT metabolism in test cultures. 

^dMTT metabolism by hyphae from PMN cultures compared to that by hyphae from PMN plus VCZ or PMN plus AmB cultures. 

^eMTT metabolism by hyphae from AmB cultures compared to that by hyphae from PMN plus AmB cultures. 

^fPMN plus VCZ was also significant (P < 0.01) compared to VCZ alone. 

Activity of VCZ with or without PMN: XTT assay.

At low VCZ concentrations and a low E/T ratio (10:1), the inhibitory activities of PMN (18%) and VCZ (32%) were more than additive when combined in culture (Table 2). Similar results were obtained in two other experiments. In a total of three experiments with 1 × 10⁵ to 3 × 10⁵ germlings/well, VCZ at 0.05 μg/ml inhibited growth of isolate 96-92 by 34% ± 5%. VCZ at 0.5 μg/ml inhibited growth by 97% under these conditions. In two experiments, isolate 92-270 was inhibited by 67% ± 7% by 0.05 μg of VCZ per ml.

TABLE 2.

Collaboration of VCZ with PMN for inhibition of A. fumigatus mycelial growth: XTT assay

Condition	VCZ (μg/ml)	ΔA410 (n = 4)a	% Inhibition	Pb
				Ic	IId	IIIe
Medium	0.00	0.477 ± 0.072	0	R
	0.01	0.388 ± 0.062	19	NS	R
	0.05	0.324 ± 0.059	32	<0.05		R
PMN (5 × 104/well)	0.00	0.391 ± 0.093	18	NS
	0.01	0.313 ± 0.066	34	<0.05	NS
	0.05	0.191 ± 0.011	60	<0.01		<0.01

^aXTT metabolism by mycelial growth in 1 h at 37°C in microtest plate wells. Inoculum, 5 × 10³ germlings/well. Values are means ± standard deviations. 

^bR, reference value; NS, not significant (P > 0.05). 

^cMetabolism of XTT by mycelia from medium (control) cultures compared with XTT metabolism by mycelia from test cultures. 

^dMetabolism of XTT by mycelia from VCZ (0.01 μg/ml) cultures compared with that of PMN plus VCZ cultures. 

^eMetabolism of XTT by mycelia from VCZ (0.05 μg/ml) cultures compared with that of PMN plus VCZ cultures. 

In an experiment with isolate 92-270 and a smaller inoculum (2 × 10³/ml), VCZ alone at 0.01, 0.05, and 0.1 μg/ml inhibited growth by 62, 72, and 86%, respectively (all P values were <0.01 compared to medium alone). At an E/T ratio of 25:1 in that experiment, PMN inhibited growth by 46% (P < 0.01) and PMN combined with VCZ produced 78, 94, and 100% inhibition, respectively, each value being significantly (P < 0.05) greater than PMN alone or the respective VCZ concentration alone. At a 400:1 E/T ratio, VCZ at 0.1 μg/ml boosted an already potent inhibition by PMN (72%) to 98% (P < 0.01). In some experiments (not shown), we found that PMN activity alone could be quite variable versus hyphae and even could be low with high E/T ratios. The variables responsible could have been the aspergillus strain, the PMN donor, and the germling size (which could vary after 24 h of incubation). However, as this study will show, even low levels of PMN activity can be boosted by VCZ, and, as detailed below, by immunostimulants.

Effect of G-CSF or GM-CSF on PMN activity and further studies of E/T ratio: XTT assay.

Treatment of PMN with G-CSF or GM-CSF during the coculture period with germlings resulted in enhanced inhibitory activity compared to control PMN (Table 3); inhibition by PMN alone (11%) increased to 40% when G-CSF (500 ng/ml) was present and to 34% when GM-CSF (500 U/ml) was present. At the same E/T ratio in other experiments, PMN inhibition of 6% significantly increased to 31 and 24% when lower doses of G-CSF (100 ng/ml) or GM-CSF (100 U/ml), respectively, were present (P was <0.05 for both).

TABLE 3.

Effect of G-CSF or GM-CSF on PMN inhibition of A. fumigatus hyphal growth: XTT assay

Condition	ΔA410 (n = 4)a	% Inhi- bition	Pb
			Ic	IId
Medium	0.745 ± 0.049	0	R
PMN (5 × 104/well)	0.663 ± 0.049	11	<0.01	R
PMN + G-CSF (ng/ml)
100	0.566 ± 0.047	24	<0.01	<0.05
500	0.445 ± 0.025	40	<0.01	<0.01
PMN + GM-CSF (U/ml)
100	0.520 ± 0.038	30	<0.01	<0.01
500	0.489 ± 0.021	34	<0.01	<0.01

^aXTT metabolism in 1 h at 37°C by hyphae from 5 × 10³ conidia/well in a microtest plate. Values are means ± standard deviations. 

^bR, reference value. 

^cMetabolism of XTT by hyphae from medium (control) cultures compared with that by hyphae of test cultures. 

^dXTT metabolism by hyphae from PMN cocultures compared with that of G-CSF- or GM-CSF-treated PMN cocultures. 

When the parameters in the experiments were changed, e.g., an E/T ratio of 25:1, PMN inhibition increased, as did inhibition by G-CSF- or GM-CSF-stimulated PMN (Fig. 3). At an E/T ratio of 400:1, inhibition by PMN alone (72%) increased to 89% when G-CSF (100 ng/ml) was present and was increased slightly by 100 U of GM-CSF per ml, significantly increasing only by 500 U of GM-CSF per ml (to 97%) (data not shown).

FIG. 3.

Effects of G-CSF and GM-CSF on antifungal activity of PMN for hyphae of A. fumigatus (isolate 96-92). Percent inhibition of hyphal growth by PMN or PMN in the presence of G-CSF (100 ng/ml) or GM-CSF (100 U/ml) is given on the vertical axis. The E/T ratio was 25:1. Data from two experiments are given. Both G-CSF-treated and GM-CSF-treated PMN produced significantly (P < 0.01) greater inhibition than did untreated PMN.

Interaction of stimulated PMN with VCZ: XTT assay.

At an E/T ratio of 10:1, where PMN activity alone was negligible, VCZ alone at 0.1 μg/ml inhibited growth by 73% and boosted inhibition by G-CSF (500 ng/ml)-treated and GM-CSF (500 U/ml)-treated PMN from 38 and 18%, respectively, to 89 and 83% (P was <0.01 for either treatment versus PMN alone).

With the XTT assay and a 50:1 E/T ratio, inhibitory activity of PMN (56%) collaborated with VCZ at 0.01 μg/ml (34% inhibition) for an additive effect on hyphal growth (84%) (Table 4). Compared to untreated PMN, G-CSF- and GM-CSF-treated PMN had significantly increased antifungal activities, of 95 and 91%, respectively. Inhibition of growth by G-CSF- or GM-CSF-treated PMN plus VCZ was significantly greater than inhibition by untreated PMN plus VCZ. Conversely, inhibition by G-CSF- or GM-CSF-treated PMN was boosted by VCZ; this was significant for GM-CSF but not G-CSF, possibly because the G-CSF-treated PMN were so inhibitory already. At an E/T ratio of 400:1, the potent PMN plus VCZ combination yielding 98% inhibition was only slightly increased by GM-CSF (to 99%), and this required 500 U/ml.

TABLE 4.

Collaboration of G-CSF- and GM-CSF-treated PMN with VCZ for activity against A. fumigatus hyphae: XTT assay

Conditiona	VCZ (μg/ml)	ΔA410 (n = 4)b	% Inhi- bition
Medium	0	0.271 ± 0.410	0
	0.01	0.180 ± 0.033	34c
PMN	0	0.119 ± 0.019	56d
	0.01	0.043 ± 0.021	84e,f
G-CSF + PMN	0	0.014 ± 0.015	95g
	0.01	0.006 ± 0.008	98h
GM-CSF + PMN	0	0.025 ± 0.020	91g
	0.01	0.002 ± 0.004	99h

^aHyphae from 10³ conidia (isolate 96-92) per microtest plate well incubated in medium ± VCZ or with PMN (5 × 10⁴ per well) ± VCZ for 24 h at 37°C. G-CSF, 100 ng/ml; GM-CSF, 500 U/ml. 

^bMetabolism of XTT by washed hyphae in 1 h at 37°C was measured spectrophotometrically. Values are means ± standard deviations. 

^cP < 0.05, compared to medium. 

^dP < 0.01, compared to medium. 

^eP < 0.05, compared to untreated PMN. 

^fP < 0.01, compared to VCZ alone. 

^gP < 0.01, compared to untreated PMN. 

^hP < 0.05, compared to PMN plus VCZ. 

Activities of VCZ and PMN with or without G-CSF or GM-CSF: MTT assay.

MTT results were consistent with the above observations. At a low E/T ratio (10:1), where PMN inhibition alone was low and variably significant, G-CSF-treated PMN collaborated with VCZ (0.5 μg/ml) for increased inhibition of hyphal growth. In this experiment, PMN were not inhibitory alone, VCZ (0.5 μg/ml) inhibited growth by 50%, and G-CSF (500 ng/ml)-treated PMN inhibited growth by 55% (P of <0.01 compared to PMN); this increased to 77% with VCZ (P of <0.05 to 0.01 versus VCZ or G-CSF PMN alone). In another experiment, VCZ inhibition was the same, GM-CSF (100 U/ml)-treated PMN inhibited growth by 61%, and the combination gave 78% inhibition.

Activity of VCZ and monocytes with or without GM-CSF: MTT assay.

Monocyte monolayers challenged with germinated conidia at a high E/T ratio (50:1) inhibited hyphal growth by 59% (Table 5). Monocyte activity is sensitive to culture conditions; with XTT assays, 96-well plates, and other times of incubation and adherence conditions, less monocyte activity was demonstrated (data not shown). VCZ alone had an effect similar to monocytes alone under the present study conditions (Table 5), i.e., inhibition of 58%. Similar results were obtained in a second, identical experiment.

TABLE 5.

Collaboration of monocytes ± GM-CSF with VCZ for inhibition of hyphal growth of A. fumigatus: MTT assay

Condition	A570 (n = 4)a	% Inhi- bition	Pb
			Ic	IId	IIIe
Medium	0.912 ± 0.471	0	R
VCZ (0.5 μg/ml)	0.380 ± 0.144	58	<0.01
Monocytes	0.374 ± 0.097	59	<0.01	R	R
Monocytes + VCZ	0.186 ± 0.096	79	<0.01	<0.01
GM-CSF (100 U/ml)
+ Monocytes	0.149 ± 0.029	83	<0.01	R	<0.01
+ Monocytes +   VCZ	0.181 ± 0.063	80	<0.01	NS

^aMetabolism of MTT by hyphae (isolate 96-92) in 3 h at 37°C. Values are means ± standard deviations from four determinations. 

^bR, reference value; NS, not significant (P > 0.05). 

^cMTT metabolism by hyphae from medium (control) cultures compared with MTT metabolism by hyphae from test cultures. 

^dMetabolism of MTT by hyphae from monocyte- or GM-CSF-treated monocyte cultures compared with respective MTT metabolism by hyphae from monocyte plus VCZ and GM-CSF plus monocyte plus VCZ cultures. 

^eMTT metabolism by hyphae from monocyte cultures compared with that by hyphae from GM-CSF-treated monocyte cultures. 

When the combination of monocytes plus VCZ was challenged with hyphae, inhibition was increased to 79%, greater than by either agent alone. GM-CSF treatment of monocytes significantly increased their inhibition of hyphal growth, from 59 to 83% (Table 5). Similar results were obtained in a second experiment. However, the combination of GM-CSF-treated monocytes and VCZ did not increase the inhibition of hyphal growth above that of GM-CSF-treated monocytes alone. In other experiments (not shown), as would be expected, G-CSF had no effect on monocyte activity.

DISCUSSION

We report here for the first time, to our knowledge, that VCZ (0.6 μg/ml) is fungicidal for conidia of A. fumigatus in vitro. These findings have implications for prophylactic therapy in patients at high risk for pulmonary aspergillosis.

Others using the broth macrodilution method (7) or the agar dilution method (10) have reported that VCZ (0.5 μg/ml) has in vitro activity against A. fumigatus hyphae. Using the MTT and XTT assays, we have confirmed these results. Moreover, we have demonstrated the utility, simplicity, and objectivity of—and data for statistical analysis provided by—the microtest plate XTT assay.

We report for the first time that under the appropriate conditions, PMN or monocytes and VCZ collaborate additively in inhibiting hyphal growth of A. fumigatus in a 24-h assay. These in vitro results may partially explain the clinical efficacy of VCZ in acute invasive aspergillosis (2).

Treatment of PMN with G-CSF has been reported to increase their activity against hyphae of A. fumigatus in a short-term assay (12). Using a 24-h coculture system with G-CSF, we have obtained similar results. In addition, we report here that GM-CSF treatment of PMN also significantly increases the inhibitory activity against hyphae of A. fumigatus. Moreover, this boost in activity due to these CSFs also significantly increases collaboration with VCZ compared to control PMN plus VCZ.

At higher E/T ratios, PMN activity is greater and the effects of the CSFs are proportionally smaller. This would suggest a greater importance of the effects of CSFs clinically in combating infection when there are less PMN available at the site of infection, i.e., in neutropenic states. Thus, the CSFs may not only increase cell numbers but may also have their greatest enhancing effects on individual cell functions at times when cell numbers are low.

Monocyte monolayers significantly inhibited hyphal growth by 59% when challenged for 24 h, and GM-CSF treatment significantly increased this to 83%. These results are similar to those reported by Roilides et al. (11), who used 4-day monocyte-derived macrophages and a 2-h challenge with germinated conidia to estimate hyphal damage. Here, we show that in 24-h combination studies, monocytes collaborated with VCZ for significantly increased inhibition of hyphal growth, from 58 to 79%. However, significantly increased inhibition of hyphal growth by a combination of GM-CSF treatment of monocytes and VCZ could not be demonstrated, possibly due to the already high inhibitory activity of GM-CSF-treated monocytes.