INTRODUCTION

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Bactericidal/permeability-increasing protein (BPI) is a cationic, 55-kDa glycoprotein found in the azurophilic granules of human polymorphonuclear leukocytes (21). It is bactericidal for a number of gram-negative bacteria and inhibits a number of alterations of normal biologic functions induced by lipopolysaccharide (LPS) (17, 18). BPI acts against bacteria by binding to LPS on the bacterial outer cell wall and disrupting the transmembrane potential (23). A recombinant fragment (rBPI₂₃), which corresponds approximately to the amino-terminal half of BPI that possesses most of the antibacterial and anti-LPS activities, was produced and was found to have the same properties as BPI (12, 22). A second form of recombinant BPI (rBPI₂₁) was derived from rBPI₂₃ by changing one of its three cysteines to alanine (13). The change resulted in improved productivity, increased stability, and reduced microheterogeneity, while it retained the bactericidal and neutralizing properties of rBPI₂₃ (13). The potent activity of rBPI₂₁ against gram-negative bacteria (11, 20) is initiated by the binding of the protein to the LPS receptor in the outer membrane, followed rapidly by an increase in the permeation of small hydrophobic molecules and irreversible cytoplasmic damage (16, 22). An initial study with rBPI₂₃, in which microtiter wells were coated with either intact or sonicated RH strain tachyzoites and increasing concentrations of ¹²⁵I-rBPI₂₃ were added, revealed that this protein bound to the whole or lysed tachyzoites of Toxoplasma gondii in a dose-dependent manner (Fig. 1). Therefore, it was considered of interest to examine the in vitro and in vivo activities of rBPI₂₁ against T. gondii when rBPI₂₁ was used alone or in combination with sulfadiazine, which is commonly used in drug combinations for the treatment of human toxoplasmosis (6, 14).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Binding of rBPI23 to immobilized tachyzoites of T. gondii.

	MATERIALS AND METHODS

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Mice. Swiss-Webster female mice (weight, 18 to 20 g at the beginning of each experiment) were purchased from Taconic Laboratories, Germantown, N.Y.

T. gondii. Tachyzoites of T. gondii RH and tissue cysts of T. gondii C56 were obtained as described previously (2). Mice were infected with 10³ tachyzoites administered intraperitoneally (i.p.) or with 10 cysts administered orally (4).

Cells. Human foreskin fibroblasts (HFFs; ATCC HS 68) were used. They were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Grand Island, N.Y.) containing 100 U of penicillin, 1 µg of streptomycin per ml, and 10% heat-inactivated fetal bovine serum (Gibco BRL) free of antibodies to T. gondii.

Drugs. rBPI₂₁ (lot 1712-970425) was prepared as described previously (12) and was formulated in 20 mM MOPS (3-[N-morpholino]propanesulfonic acid), 150 mM NaCl, 0.2% Pluronic P103 (BASF, Parsippany, N.J.), and 0.35% EDTA. A similar buffer without rBPI₂₁ was used for all dilutions and controls. Sulfadiazine was purchased from Sigma Chemical Co., St. Louis, Mo.

In vitro experiments. (i) Effect of rBPI₂₁ on extracellular tachyzoites. To determine whether exposure of extracellular tachyzoites to rBPI₂₁ would affect their viability or capacity to invade cells, they were harvested from the peritoneal cavities of mice infected for 2 days, washed by centrifugation with DMEM, resuspended in DMEM, counted with a hemocytometer, and distributed into sterile glass test tubes. The washed tachyzoites were exposed to increasing concentrations of rBPI₂₁ of 0, 0.5, 5.0, or 25.0 µg/ml for 10, 30, or 60 min in a CO₂ incubator at 37°C. Parasites treated with DMEM or dilution buffer served as controls. At the end of the incubation, tachyzoites were sedimented by centrifugation, the medium was aspirated, and the organisms were resuspended in 250 µl of phosphate-buffered saline (pH 7.2). The viability of the tachyzoites was determined by microscopic examination after the addition of a solution of methylene blue to the parasite suspension. Nonviable tachyzoites appear thin and distorted and do not take up the stain, whereas viable parasites appear round and stain deep blue. The ability of tachyzoites to invade cells after treatment with rBPI₂₁ was examined by exposing extracellular tachyzoites to the drug as described above. Thereafter, the tachyzoites were washed by centrifugation to remove the drug, resuspended in culture medium, and placed on monolayers of HFFs in tissue culture slides (Lalge Nunc International, Naperville, Ill.). Following a 24-h incubation at 37°C in a 5% CO₂ environment, the monolayers were fixed, stained with DiffQuick (Baxter, McGaw Park, Ill.), and examined microscopically to determine the percentage of infected cells compared with the percentage of infected control cells.

(ii) Effect of rBPI₂₁ on intracellular replication of T. gondii. rBPI₂₁ was used at concentrations of 0, 0.05, 0.5, 5.0, and 25.0 µg/ml. Dilutions of rBPI₂₁ and sulfadiazine were made in DMEM. In the studies performed to determine the synergistic activity of the combination of rBPI₂₁ and sulfadiazine, the drugs were examined at concentrations ranging from 0.6 to 13.8 µg/ml at their equipotent ratio of 1:1.5. This ratio was based on their 50% inhibitory concentrations (IC₅₀s), which were determined in preliminary experiments. To determine a dose-response for each drug alone, the activities of rBPI₂₁ and sulfadiazine were evaluated at concentrations from 0.6 to 10.0 µg/ml and 1.0 to 15.0 µg/ml, respectively. In vitro activity was defined as the capacity of the drug to inhibit the intracellular replication of T. gondii and was determined by a modification of the previously described [³H]uracil incorporation technique (3). Briefly, HFF cells were plated at 10⁴ cells/well in 96-well flat-bottom tissue culture microtiter plates, and the plates were incubated at 37°C in a 5% CO₂ incubator. After confluence, the monolayers were infected with tachyzoites at a ratio of 3 tachyzoites/cell (approximately 6 × 10⁴ tachyzoites/well). At 4 h after infection, the monolayers were washed to remove free parasites and various concentrations of rBPI₂₁, sulfadiazine, or the drug combination were added for the duration of the experiment. Triplicate wells were used for each drug concentration. The time of addition of the drugs to the wells marked the starting time point. At 4 h prior to the harvesting of the cells, [5,6-³H]uracil (1 µCi/well) was added to each well and its incorporation was determined at 24, 48, and 72 h following the addition of the test drug. The cells were collected with a cell harvester, and radioactivity was counted with a scintillation counter. Infected monolayers treated with medium that contained the respective drug diluent alone served as controls. Results are reported as the level of incorporation of [5,6-³H]uracil in the treated monolayers compared with that in untreated control monolayers. Quantification was achieved by calculating the percentage of [5,6-³H]uracil incorporated by replicating tachyzoites in treated cultures compared with the percentage incorporated in untreated control cultures. Combination analysis was performed with computer software for dose-effect analysis (CalcuSyn; Biosoft, Ferguson, Mo.) that is based on median-effect principles (9). To assess the antitoxoplasma effects of combinations of these two drugs, combination indices (CIs) (9) or isobologram plots (7) were used. CIs of <1, 1, or >1 indicate synergism, an additive effect, or antagonism, respectively. CIs of 0.85 to 0.90 represent moderate synergism and CIs of 0.70 to 0.85 represent slight synergism, as described by Chou and Hayball (8). In isobologram plots, datum points below the iso-effect line, on the line, or above the line indicate synergism, an additive effect, or antagonism, respectively (7).

In vivo experiments. rBPI₂₁ was used at doses of 5, 10, or 20 mg/kg of body weight and was administered by i.p. injection as a single daily dose for 10 days beginning 24 h after infection with tachyzoites or with tissue cysts. Previous experiments determined that the doses of sulfadiazine required to cause only a prolongation of time to death or to protect fewer than 20% of mice infected i.p. with strain RH tachyzoites or orally with strain C56 cysts against death were 150 and 15 mg per liter of drinking water, respectively. This difference in dosages is due to the different virulences of each strain of T. gondii for mice, the different routes of infection, and the different pathologies. Treatment with sulfadiazine was also initiated 24 h after infection and was continued for 10 days. Controls were treated with rBPI₂₁ alone, the diluent of rBPI₂₁, or sulfadiazine alone. The surviving mice were examined for residual infection with T. gondii by i.p. injection of portions of triturated liver and spleen into mice or by microscopic examination of triturated brain tissue (5).

Statistical analysis. The in vitro effects of the drugs used in combination were analyzed by the median-effect principles as described by Chou and Talalay (9). The P values for the in vivo effects of the drugs used in combination were obtained by the log-rank test of the Kaplan-Meier survival analysis. P values of 0.05 were considered statistically significant.

	RESULTS

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In vitro experiments. In two experiments, treatment of extracellular tachyzoites with rBPI₂₁ did not result in a loss of viability, as determined by the methylene blue staining method. In addition, the capacity of tachyzoites to invade and replicate within cells was not altered by prior treatment with rBPI₂₁.

View larger version (38K): [in this window] [in a new window]   FIG. 2.   Effect of rBPI21 on intracellular replication of tachyzoites in vitro as determined by [3H]uracil incorporation (A) and on the metabolic activity of the host HFF cells as determined by the MTT assay (B). Results are presented as means + standard deviations from triplicate assays.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Effects of rBPI21, sulfadiazine, or their combination at a ratio of 1:1.5 on inhibition of the intracellular replication of tachyzoites in vitro represented as fractional effect; 1 is equal to 100% inhibition (A). The combined effect of rBPI21 and sulfadiazine was evaluated by the CI (B) and isobologram (C) methods. CIs of 0.70 to 0.85, 0.85 to 0.90, 0.90 to 1.10, or >1.10 indicate moderate synergism, slight synergism, additive effect, or antagonism, respectively. In the isobologram plots, points below the line, on the line, or above the line indicate synergism, an additive effect, or antagonism, respectively. ED50, ED75, and ED90, 50, 75, and 90% effective doses, respectively.

In vivo experiments. In two separate experiments with 10 mice per group, 100% of mice infected orally with cysts of strain C56 and not treated or treated with diluent died by day 16 of infection, whereas mice treated i.p. with rBPI₂₁ alone at 10 or 20 mg/kg/day had survival rates of 10 and 20%, respectively (Fig. 4A). However, survival analysis with these groups of mice did not reveal significant differences between the treated and the control groups of mice. All mice infected i.p. with strain RH tachyzoites and not treated or treated with diluent died by day 9 of infection (Fig. 4B). In contrast, infected mice treated with dosages of rBPI₂₁ of from 0.5 to 10 mg/kg/day had significantly prolonged times to death (P = 0.0023) compared with those for the untreated controls. Infected mice treated with rBPI₂₁ at 20 mg/kg/day had a 20% survival rate (Fig. 4B).

View larger version (25K): [in this window] [in a new window]   FIG. 4.   Effect of treatment of mice infected orally with cysts of T. gondii C56 (A) or with tachyzoites of T. gondii RH (B) with rBPI21 administered i.p.

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Effect of treatment of mice infected orally with cysts of T. gondii with C56 rBPI21 at 10 (A) or 20 (B) mg/kg/day alone or in combination with 15 mg of sulfadiazine (Sulfa) per liter. rBPI21 and sulfadiazine were administered i.p. and in drinking water, respectively. All the control mice had died by day 16 postinfection. Although prolongation of time to death was noted in the mice treated with the combination, the differences between this group and the groups treated with each drug alone were not statistically significant.

View larger version (23K): [in this window] [in a new window]   FIG. 6.   Effect of treatment of mice infected i.p. with tachyzoites of T. gondii RH with 5 (A), 10 (B), or 20 (C) mg of rBPI21 per kg/day alone or in combination with 150 mg of sulfadiazine (Sulfa) per liter. rBPI21 and sulfadiazine were administered i.p. and in drinking water, respectively. All the control mice had died by day 12 postinfection. The difference in the survival rates between the groups treated with each drug alone and the group treated with the combination was statistically significant (P = 0.001) for each of the combinations.

	DISCUSSION

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The results presented above indicate that rBPI₂₁ inhibits the intracellular replication of T. gondii within HFFs, prolongs survival in mice, and protects mice against death due to acute toxoplasmosis. This is the first report of the activity of rBPI₂₁ against a protozoan parasite. The effect of rBPI₂₁ against T. gondii was enhanced, particularly in the murine model, when it was used in combination with a dose of sulfadiazine that was not protective when the sulfadiazine was used alone. The enhancement of antibiotic activity by rBPI₂₁ has been reported previously in mice, rabbits (15), and baboons (19) infected with Escherichia coli and treated with either cefamandole (1) or cefotaxime (19) in combination with rBPI₂₁.

In our experiments, the significant protective activity of rBPI₂₁ was noted in the murine model of toxoplasmosis in which both infection and treatment with rBPI₂₁ were by the i.p. route. When infection was with T. gondii cysts by the oral route and treatment was administered i.p., prolongation of time to death and survival were observed, but the difference between treated groups and controls did not attain statistical significance. The reason for this difference in activity of rBPI₂₁ in two models of acute toxoplasmosis is not clear. However, differences in the pathogenicities of the two strains of T. gondii, in the routes of infection, and in the dosages of drugs used may have contributed to the observed differences. As is true for almost every antibiotic that has been shown to have activity in vitro and in vivo against T. gondii, it is unclear whether the in vitro effect of rBPI₂₁ is on the parasite or the host cells, or both, or whether it is additionally on the immune system of the mice in vivo. In addition, direct contact between rBPI₂₁ and tachyzoites did not cause a loss of viability of the organisms and did not impair their capacity to invade cells. Thus, direct contact with the parasite may not be necessary for the inhibitory effect of rBPI₂₁ on T. gondii. A number of investigators (2, 10) have demonstrated that BPI inhibits the ability of LPS to stimulate the production of cytokines such as tumor necrosis factor alpha and interleukin-6 by peripheral blood mononuclear cells. BPI also suppressed LPS-dependent NO secretion by mouse macrophages. This raises the possibility that certain of the effects that we observed with rBPI₂₁ may have resulted from an additional effect of rBPI₂₁ on cytokine production in vivo.

The remarkable synergistic effect of rBPI₂₁ and sulfadiazine in the treatment of acute murine toxoplasmosis is of interest because therapy of acute toxoplasmosis and/or toxoplasmic encephalitis still presents problems, particularly in severely immunocompromised individuals. In these individuals, single-drug therapy is often ineffective and combination-drug treatment with currently available regimens is often associated with untoward side effects (14). Our results suggest that rBPI₂₁, when used in combination with other drugs, may be useful for the treatment of toxoplasmosis in humans and perhaps infections caused by other nonbacterial pathogens as well.