Aprotozoan parasite, Toxoplasma gondii, has been recognized. Effects of Sulfamethoxazole on Murine Ocular Toxoplasmosis in Interferon- Knockout Mice

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1 Effects of Sulfamethoxazole on Murine Ocular Toxoplasmosis in Interferon- Knockout Mice Kazumi Norose, 1 Fumie Aosai, 1 Hye-Seong Mun, 1 and Akihiko Yano 1,2 PURPOSE. To evaluate the effects of sulfamethoxazole (SMX) on experimental ocular toxoplasmosis by quantitative competitive polymerase chain reaction (QC-PCR) assay. METHODS. Wild-type (WT) C57BL/6 and WT BALB/c mice and interferon- knockout (GKO) mice were infected orally with Toxoplasma gondii of the Fukaya strain. Mice were classified into groups. The first group (G1) remained untreated, the second group (G2) had a short SMX treatment period, and the third group (G3) received treatment continuously. WT and GKO mice were divided into G1 and G3, and G1, G2, and G3, respectively. T. gondii burdens were evaluated by QC-PCR assay. The effect on stage distribution was analyzed by reverse transcription-pcr. RESULTS. SMX significantly decreased mortality among the infected WT C57BL/6 and GKO mice. In WT G1 mice, T. gondii DNA was detected in all organs and tissues, although in G3 mice it was detected only in the brain. In GKO C57BL/6 G1 mice, the protozoan proliferated much more actively than in the WT mice. In the GKO C57BL/6 G2 mice, the number of T. gondii was less than in G1 during the treatment, although the protozoan reappeared after cessation of treatment. In GKO C57BL/6 G3 mice, T. gondii DNA was detected in the brain, optic nerve, and retina, but not in the iris, choroid, sclera, and blood. In GKO BALB/c mice, the patterns of the kinetics of protozoan abundance in various organs were similar or were milder than those in GKO C57BL/6 mice. In SMX-treated GKO mice, the percentage of bradyzoites increased and that of tachyzoites decreased in the organs and tissues. CONCLUSIONS. SMX decreased the parasitic load in both WT and GKO mice. SMX decreased the tachyzoite load but did not completely eliminate bradyzoites in GKO mice. The present mouse model was used successfully to assess treatment effects in a quantitative fashion. (Invest Ophthalmol Vis Sci. 2006;47: ) DOI: /iovs Aprotozoan parasite, Toxoplasma gondii, has been recognized as a human ocular pathogen in both immunocompetent and immunocompromised individuals. 1 4 From the 1 Department of Infection and Host Defense, Graduate School of Medicine, Chiba University, Chiba, Japan. 2 Deceased. Supported in part by Grants , , , , and for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and the Japanese Science Promotion Society. Submitted for publication June 14, 2005; revised August 12 and September 11, 2005; accepted November 21, Disclosure: K. Norose, None; F. Aosai, None; H.-S. Mun, None; A. Yano, None The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked advertisement in accordance with 18 U.S.C solely to indicate this fact. Corresponding author: Kazumi Norose, Department of Infection and Host Defense, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuou-ku, Chiba, , Japan; norose@faculty.chiba-u.jp. Treatment of the disease in immunocompetent hosts is problematic for ophthalmologists because ocular toxoplasmosis is usually a self-limiting disease, even without treatment, especially in the case of small, peripheral retinal lesions. Furthermore, currently available drugs do not eliminate tissue cysts and therefore cannot prevent reactivation. In a systematic review of the medical literature, Stanford et al. 5 identified only three prospective, randomized, placebo-controlled clinical trials for the treatment of ocular toxoplasmosis in immunocompetent patients, all of which were methodologically poor, with two of them being performed more than 40 years ago. They concluded that no properly designed studies have shown the effectiveness of antibiotics in ocular toxoplasmosis. 5 Their review should not be interpreted to mean that treatment has no effect, however. Although it has been difficult to demonstrate that treatment alters the natural course of the active disease, there has been evidence of the effects of treatment at an observational level. 6 8 Rothova et al. 6 found, in a nonrandomized study, a reduction in the size of retinal inflammatory lesions of pyrimethamine-treated patients compared with untreated patients, although no difference in the duration of inflammatory activity was observed between them. Silveira et al. 7 reported that long-term intermittent treatment with trimethoprim/sulfamethoxazole can reduce the rate of recurrent toxoplasmic retinochoroiditis. A physician survey study 9 in 2001 showed that 15% treated all cases regardless of clinical findings (in contrast to 6% in ), and that a total of nine drugs were used as the treatment of choice for typical cases of recurrent toxoplasmic retinochoroiditis, with the combination of pyrimethamine, sulfadiazine, and prednisone being the most commonly used regimen. They concluded that there is still no consensus regarding the choice of antiparasitic agents for treatment regimens. In contrast, with the spread of acquired immune deficiency syndrome (AIDS), the frequency of severe disseminated toxoplasmosis in humans has risen. 11 Obviously, there is a need for better prophylaxis and for a means to eradicate the parasite from body tissues. Evaluation of treatment regimens for human toxoplasmic retinochoroiditis is made especially difficult because of ethical considerations and the unavailability of retinal biopsy. 5 Moreover, inherent differences between humans and animals can reduce the relevance of data obtained experimentally, although animal models have greatly improved our knowledge of various aspects of toxoplasmosis. In experimental ocular toxoplasmosis, animals acutely infected with an inoculation of an avirulent or weakly virulent Toxoplasma strain via intraperitoneal 12 or intraocular 13 injection (i.e., via nonnatural infection routes), have been treated with an agent (or agents), and the disease has been evaluated by clinical observations such as ophthalmoscopy and slit lamp, 12,13 cerebral and retinal cyst counts under the microscope, 12 histopathological findings, 13 and isolation of Toxoplasma from the retina or choroid. 13 A simpler, more rapid and sensitive system to quantitate protection would greatly facilitate antimicrobial therapy studies. For this purpose, we developed a system to quantitate T. gondii load using quantitative competitive polymerase chain reaction (QC-PCR) amplification of DNA obtained from the eyes of wild-type (WT) and interferon- knockout (GKO) mice, Investigative Ophthalmology & Visual Science, January 2006, Vol. 47, No. 1 Copyright Association for Research in Vision and Ophthalmology 265

2 266 Norose et al. IOVS, January 2006, Vol. 47, No. 1 recently described as a reliable animal model for Toxoplasma retinochoroiditis 14 in immunocompromised hosts that were infected with avirulent cyst-forming T. gondii perorally and treated with sulfamethoxazole (SMX; Shionogi Co., Ltd., Osaka, Japan). MATERIALS AND METHODS Toxoplasma gondii Cysts of an avirulent Fukaya strain of T. gondii were obtained as previously described. 15 Experimental Animals Eight- to 10-week-old inbred WT C57BL/6 and WT BALB/c mice were purchased from SLC (Hamamatsu, Japan). GKO mice were a generous gift from Yoichiro Iwakura (Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan). The creation of C57BL/6 and BALB/c mice lacking the Ifn-G gene and the methods for genotyping have been described in detail elsewhere. 16 Mice were housed in accordance with Chiba University guidelines and were bred in the animal center of Chiba University. Age- and sexmatched GKO mice were used. All mice had normal physical and ophthalmic examinations and had no detectable serum antibodies to T. gondii before the present infection. Induction of Toxoplasmosis in Mice All experiments in this study were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All mice were infected perorally with 10 cysts of Fukaya strain administered with a syringe fitted with a 19-gauge, round-ended needle on day 0 of the experiment. WT mice were classified into two groups: (1) untreated (G1) and (2) treated continuously from day 4 after infection (PI) through day 28 PI (G3). G1 and G3 mice were killed at 0, 5, 7, 10, 14, 21, and 28 days PI. GKO mice were classified into three groups: (1) untreated and killed at 0, 5, 7, and 10 days PI (G1); (2) treated from day 4 until day 15 PI and then left untreated and killed at 0, 5, 7, 10, 15, 18, and 22 days PI (G2); (3) treated continuously from day 4 through day 28 PI and killed at 0, 5, 7, 10, 15, 18, 22, and 28 days PI (G3). Survival of the mice was monitored daily, and cumulative mortality was calculated. The experiments were performed three times. For survival, the total number of each group was 10. For QC-PCR, the total number of G1 and G3 of C57BL/6 WT mice were 50 and 20, respectively, and those of BALB/c WT mice were 25 and 20, respectively. Those of G1, G2, and G3 of both strains of GKO mice were 12, 20, and 25, respectively. For RT-PCR, four GKO C57BL/6 mice were used for each experimental group (G1 and G2). Treatment with SMX SMX was administered ad libitum in drinking water at a dose of 1 mg/ml from day 4 PI. SMX was dissolved first in 2 N NaOH to facilitate its absorption in the water. ph was then adjusted to 7 with 10 N HCl. A 6-mL average daily fluid consumption of mice was continued under laboratory conditions. QC-PCR Genomic DNA (gdna) from the respectively pooled brain, optic nerve (ON), posterior retina, peripheral retina, iris, choroid, sclera, and blood from three mice of the same group was prepared as previously described. 14 The retina was cut along the center of the distance from the ON head and ora serrata, separating the posterior and peripheral retina, respectively. Using 1 g of gdna from these organs and tissues, QC-PCR was performed to determine the distribution of T. gondii as described previously. 17 Briefly, gdna (1 g) extracted from these organs and tissues was coamplified in the reaction buffer containing 10 M bovine serum albumin with a constant concentration of truncated SAG1 (major surface antigen of T. gondii) DNA that competitively binds oligo primers with WT SAG1. The amplified cdnas were electrophoretically separated on 1% agarose gel containing ethidium bromide, and the ratio to competitor (T/C) SAG1 DNA subsequently amplified was measured with a gel densitometer (IPLab; Signal Analytical Corp., Vienna, VA). The number of T. gondii was calculated as described previously. 17 The experiments were repeated three times. Reverse Transcription-PCR for Analysis of Stage Distribution of T. gondii To study the effects of SMX on stage distribution in vivo, we investigated the expressions of SAG1 (as a marker of tachyzoites 18 ), and T.g.HSP30/bag1 (as a marker of bradyzoites 19 ) mrnas by RT-PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. mrna from the separately pooled brain, ON, posterior retina, peripheral retina, iris, choroid, and blood from four mice of the same group was prepared. For RT-PCR analysis, GKO C57BL/6 mice were killed at 10 days PI. The expression levels of SAG1 and T.g.HSP30/bag1 mrnas, as well as the percentage of expression levels of these mrnas were calculated from the densities of their RT-PCR products using the following formula: mrna expression and % mrna expression SAG1 mrna, or T.g.HSP30/bag1 mrna GAPDH mrna SAG1 mrna, or T.g.HSP30/bag1 mrna 100. SAG1 mrna T.g.HSP30/bag1 mrna The experiment was repeated three times. Primers The primers used were as follows: for SAG1 forward (5 -TCG GAT CCC CCT CTT GTT GC-3 ), corresponding to nucleotides of the T. gondii SAG1 gene, and reverse (5 -CTC CAG TTT CAC GGT ACA GT-3 ), corresponding to nucleotides ; for T.g.HSP30/bag1, forward (5 -GGG AAT TCA TGG CGC CGT CAG CAT C-3 ) and reverse (5 -GGG CGG CCG CCT ACT TCA CGC TGA TTT GTT-3 ); for GAPDH, forward (5 -ACC ACA GTC CAT GCC ATC AC-3 ) and reverse (5 -TCC ACC ACC CTG TTG CTG TA-3 ). Statistics The significance of differences in the survival between groups was determined by the Kaplan-Meier method. The significance of differences in T. gondii load and stage distribution was determined by Mann-Whitney test. Data were considered significant at P RESULTS Mortality Sixty percent of untreated WT C57BL/6 mice (susceptible strain) died during the 28-day observation period (Fig. 1a), whereas all treated WT C57BL/6 mice survived (P 0.05). WT BALB/c mice (resistant strain) survived even without treatment. On the contrary, untreated GKO C57BL/6 and BALB/c mice died within 10 to 11 days PI, whereas treated GKO mice survived under continuous treatment (Fig. 1b; P 0.05). In addition, GKO mice died within 10 days after cessation of the therapy (P 0.05). Influence of SMX on the Parasite Loads of WT Mice Infected Perorally with T. gondii The time course of protozoan proliferation in different organs and tissues of untreated WT mice (G1) varied (Fig. 2). The

3 IOVS, January 2006, Vol. 47, No. 1 Sulfamethoxazole and Ocular Toxoplasmosis 267 abundance reaching a peak at 14 to 21 days PI and decreasing thereafter. In treated WT BALB/c mice (G3), the protozoan DNA was not detected from all organs and tissues we examined except the brain, where the T. gondii number decreased after the treatment. Influence of SMX on the Parasite Loads of GKO Mice Infected Perorally with T. gondii In untreated GKO C57BL/6 mice (G1) on day 7 PI (Figs. 3a h), the kinetics of T. gondii loads showed that it was detected in all organs and tissues examined except the peripheral retina. The pattern of T. gondii loads in organs and tissues under this treatment could be classified into two groups. The first group included the brain, ON, and posterior and peripheral retinas (Figs. 3a d, respectively), from which the parasite DNA was detected during continuous SMX treatment. T. gondii abundance was the highest (P 0.05) in the ON after continuous treatment. The second group of organs and tissues included the iris, choroid, sclera, and blood (Figs. 3e h, respectively). The protozoan DNA was not detected from these organs and tissues under continuous SMX treatment. The protozoan began to proliferate after the treatment was stopped in both groups (G2). In untreated GKO BALB/c mice (G1), the kinetic patterns of the abundance of protozoan in the organs and tissues were similar to those in untreated GKO C57BL/6 mice (Figs. 3i p). T. gondii DNA was not detected in any of these organs and tissues except the brain under continuous SMX treatment. It should be noted that even in the brain, the T. gondii load tended to decline under continuous treatment, but the protozoan began to proliferate after its cessation. FIGURE 1. Survival rate. (a) WT C57BL/6 G1 ( ), WT C57BL/6 G3 (Œ), WT BALB/c G1 ( ), and WT BALB/c G3 ( ). Horizontal bar beneath the x-axis indicate the duration of the treatment in G3. *Significant difference at P 0.05 between G1 and G3. (b) GKO C57BL/6 G1 ( ), GKO C57BL/6 G2 (f), GKO C57BL/6 G3 (Œ), GKO BALB/c G1 ( ), GKO BALB/c G2 ( ), and GKO BALB/c G3 ( ). The experiments were performed three times, with similar results. Horizontal short and long bars beneath the x-axis indicate the duration of treatments in G2 and G3, respectively. *Significant difference at P 0.05 between G1 and G2 and between G2 and G3. kinetics of T. gondii loads showed that in the susceptible strain WT C57BL/6 (Figs. 2a e), T. gondii was detected first in the blood, and second in the brain, ON, posterior and peripheral retinas, 7 and 10 days PI, respectively. Although the number of protozoa in the brain, ON, posterior and peripheral retinas continued to increase gradually, that in blood was at its maximum 7 days PI and decreased thereafter. The protozoan DNA was not detected from the ON, posterior and peripheral retinas, and blood of G3 by QC-PCR, and in the brain the T. gondii number was significantly decreased (P 0.05) after the treatment (G3). In the resistant strain WT BALB/c, the abundance of T. gondii in organs and tissues such as blood 7 days PI and brain, posterior and peripheral retinas 28 days PI of untreated mice (G1) was much less (P 0.05) than in WT C57BL/6 mice (Figs. 2f j). T. gondii was first detected in the blood 7 days PI, decreasing thereafter, a pattern similar to that in WT C57BL/6 mice. The protozoan was then detected in the brain, ON, posterior and peripheral retinas at 10 to 14 days PI, with their Influence of SMX on Stage Distribution In the brain, ON, and posterior retina of untreated GKO C57BL/6 mice (G1), the SAG1/GAPDH mrna expression was significantly lower (P 0.05) on day 10 PI than the T.g.HSP30/ bag1/gapdh mrna expression (Table 1). In contrast, the SAG1/GAPDH mrna expression was higher on day 10 PI than the T.g.HSP30/bag1/GAPDH mrna expression in the peripheral retina, iris, choroid, and blood, although the differences were not significant except in the blood. In all organs and tissues of SMX-treated mice (G2), SAG1 mrna expression downregulation was induced by SMX, with only T.g.HSP30/bag1 mrna being expressed in these organs and tissues, and the differences were significant (P 0.05). SAG1/GAPDH mrna expression in the brain, ON, and blood and T.g.HSP30/bag1/GAPDH mrna expression in the brain and ON of G2 was significantly lower (P 0.05) than in G1. DISCUSSION In this study we developed a sensitive detection system that can provide effective and efficient measurement of the ability of antimicrobial therapy to provide protection against T. gondii infection to the eye. SMX alone was chosen as an antitoxoplasma drug for this study on the basis of its convenience, low cost, and low rate of side effects. In addition, this is the first step in the experimental assessment study of the use of this drug alone, not in combination. The frequency of administration of trimethoprim/smx, which has been used widely in patients with AIDS for treatment and prevention of toxoplasmic encephalitis 9,20 and was used by more uveitis specialists for typical ocular toxoplasmosis in than in 1991, 10 has been chosen empirically. There are many laboratory and clinical reports suggesting that SMX alone or in combination with other drugs is effective against T. gondii. 7,20 22

4 268 Norose et al. IOVS, January 2006, Vol. 47, No. 1 FIGURE 2. Kinetics of T. gondii abundance in organs and tissues of WT C57BL/6 (a e) and BALB/c (f j) mice. WT C57BL/6 G1 ( ), WT C57BL/6 G3 (Œ), WT BALB/c G1 ( ), and WT BALB/c G3 ( ). (a, f) brain; (b, g) ON; (c, h) posterior retina; (d, i) peripheral retina; and (e, j) blood. Data are expressed as the mean number of parasites per microgram of specimen DNA SD. Experiments were performed three times, with similar results. *Significant difference at P 0.05 between G1 and G3; Significant difference at P 0.05 between the same organs and tissues of C57BL/6 and those of BALB/c mice, in which the blood and other organs and tissues were compared at 7 days and 28 days PI, respectively. Horizontal bars beneath the x-axis indicate the duration of treatments in G3. Our present results clearly showed that SMX decreased the parasitic loads of T. gondii in experimental ocular toxoplasmosis in WT and GKO mice, used as models of immunocompetent and immunocompromised hosts, respectively. Sulfonamides have been used for ocular toxoplasmosis for some 50 years, since Eyles and Coleman 23 in 1953 demonstrated the synergistic action of pyrimethamine and sulfonamides on T. gondii. Sulfonamides interfere with the formation of folic acid from para-amino benzoic acid. As a result, errors in nuclear division occur during parasitic replication. Sulfadiazine protected all mice infected intraperitoneally with the RH strain, and decreased the parasitic load in the blood, lungs, and brain. 24 Miédougé et al. 25 reported that brain and lung parasitic loads were significantly increased in mice infected with avirulent Beverley strain cysts and treated with anti-interferon, and that this increase was prevented by the concomitant administration of pyrimethamine and sulfadiazine, suggesting that early prophylaxis would be suitable. Remington 21 treated mice infected with the RH strain of T. gondii intraperitoneally with trimethoprim and SMX, and reported that only SMX alone at higher dosages was able to kill the RH strain of T. gondii and cure the animals. Sulfonamides vary in their antitoxoplasmic activity. Unlike SMX and sulfadiazine, sulfisoxazole was found to be ineffective in mice inoculated with T. gondii RH strain. 26 Pharmacokinetic differences might account for these discrepancies, since SMX and sulfadiazine have a prolonged serum half-life (12 18 hours) compared with sulfisoxazole (3 6 hours). 27 The 50% inhibitory concentrations (IC 50 ) of sulfadiazine, SMX, and sulfametrole are all within the same range. 22 The management of ocular toxoplasmosis in immunocompetent adults is based on the following principles: (1) the active phase of the disease is self-limiting; (2) retinal necrosis is due to proliferation of organisms; (3) the immune response to these organisms can result in additional damage to intraocular tissues; and (4) currently available drugs cannot eliminate tissue cysts and therefore cannot prevent recurrences. 28 In view of the present study that SMX decreased the parasitic loads in WT mice, treatment of ocular toxoplasmosis even in immunocompetent hosts would be recommended to reduce the possibility of retinal damage. Treatment may be extremely important in specific situations, such as in pregnant women, newborns, and immunosuppressed patients. 28 Our present data suggest that SMX treatment should be continued much longer in immunocompromised hosts than in immunocompetent hosts, because the parasite DNA was detected in the brain, ON, and retinas of GKO C57BL/6 mice and in the brain of GKO BALB/c mice even under continuous treatment. Of course, careful monitoring of possible side effects of the drug will be imperative. Although there are many animal studies for determining the efficiency of drug therapy in toxoplasmosis, 21,25,26,29 31 limited animal studies 12,13 are available for ocular toxoplasmosis, and the results obtained have varied. Tabbara et al. 13 reported that, in light of the clinical improvement, isolation of Toxoplasma organisms, and histologic examinations, clindamycin was effective in the treatment of experimental toxoplasmic retinochoroiditis in rabbits injected in the suprachoroidal space with the Beverley strain of T. gondii. Gormley et al. 12

5 IOVS, January 2006, Vol. 47, No. 1 Sulfamethoxazole and Ocular Toxoplasmosis 269 FIGURE 3. Kinetics of T. gondii abundance in organs and tissues of GKO C57BL/6 (a h) and GKO BALB/c (i p) mice. GKO C57BL/6 G1 ( ), GKO C57BL/6 G2 (f), GKO C57BL/6 G3 (Œ), GKO BALB/c G1 ( ), GKO BALB/c G2 ( ), and GKO BALB/c G3 ( ). (a, i) brain; (b, j) ON; (c, k) posterior retina; (d, l) peripheral retina; (e, m) iris; (f, n) choroid; (g, o) sclera; and (h, p) blood. Data are expressed as mean number of parasites per microgram of specimen DNA SD. Experiments were performed three times, with similar results. *Significant difference at P 0.05 between G1 and G2 and between G2 and G3; significant difference at P 0.05 between organs and tissues of the same group. Horizontal short and long bars beneath the x-axis indicate the duration of treatments in G2 and G3, respectively. evaluated the effects of drug therapies (pyrimethamine combined with sulfadiazine; clindamycin; spiramycin; atovaquone) on the clinical course of acute acquired Toxoplasma retinochoroiditis and on the number of Toxoplasma cysts present in the brain and ocular tissues in a hamster animal model infected intraperitoneally with an avirulent ME49 cyst-forming strain of T. gondii. They showed that none of the drugs administered altered the course of the acute disease, judged by clinical examination, and that atovaquone alone significantly reduced the number of cerebral Toxoplasma cysts after acute and chronic disease. Our present study showed that, in continuously SMXtreated GKO C57BL/6 mice, T. gondii DNA was still detected by QC-PCR in the brain, ON, and posterior retina in which bradyzoites were predominant on day 10 PI. Furthermore, in tachyzoite-predominant tissues or organs such as the peripheral retina, iris, choroid, and blood on day 10 PI, we could not detect parasite DNA by QC-PCR after 24 days of continuous

6 270 Norose et al. IOVS, January 2006, Vol. 47, No. 1 TABLE 1. Expressions of SAG1 mrna/gapdh and T.g.HSP30/bag1 mrna/gapdh in GKO C57BL/6 Mice after Peroral Infection with 10 T. gondii Cysts Expression (%) Tissue Group SAG1/GAPDH T.g.HSP30/bag1/GAPDH Brain G * ( ) ( ) G * ( ) ( ) Optic nerve G * ( ) ( ) G * ( ) ( ) Posterior retina G * ( ) ( ) Peripheral retina G ( ) ( ) Iris G ( ) ( ) Choroid G ( ) ( ) Blood G * ( ) ( ) G * ( ) ( ) Expression was investigated at day 10 PI in untreated GKO (G1) and treated (G2) mice. The experiment was repeated 3 times. Data are expressed as the mean SD. ND, not done because protozoan DNA was not detected from these organs by QC-PCR. Percent expression, indicated as the mean % expression SD, is shown in parentheses beside each expression. * Significant difference at P 0.05 between mean expression of SAG1/GAPDH and T.g.HSP30/bag1/GAPDH in the same group and tissue. Significant difference at P 0.05 between G1 and G2 of mean expression of SAG1/GAPDH or T.g.HSP30/bag1/GAPDH. treatment except peripheral retina. Thus, there were organ or tissue differences in the efficacy of SMX against the parasite loads in this study. It is well known that chemotherapy, including that with SMX, acts on the proliferative stage of T. gondii (i.e., tachyzoites), but not on the cyst-forming stage (i.e., bradyzoites 32 ), although experimental studies suggest that atovaquone 12,31 and azithromycin 30,31 affect bradyzoites, either by reducing the cyst burden or causing morphologic change in the bradyzoite itself. Bradyzoites are characterized by their resistance to both the immune system and chemotherapy. We detected only bradyzoites on day 10 PI in the brain, ON, and blood of GKO C57BL/6 mice that had been treated from day 4 PI. The stimulus that triggers Toxoplasma encystation and the molecular mechanisms triggering the switch from tachyzoite to bradyzoite still remain unknown. 33 Elucidation of these mechanisms is eagerly awaited, since bradyzoites within tissue cysts are not only the source of infection transferred from domestic animals to humans and of reactivation of a local retinal or brain tissue cyst, but can also be converted to tachyzoites that are the cause of fatal toxoplasmic encephalitis in AIDS patients. The evaluation of the potential risk of reactivation of toxoplasmosis will be the subject of future studies of immunosuppressive drugs 34 or lethal irradiation. We demonstrated in a previous study 14 that differences existed not only in the number of parasites but also in their distribution in the various eye parts between WT and GKO mice. In the present study, we showed that there were genetic differences that determined the efficacy of SMX against parasite loads between C57BL/6 and BALB/c WT mice and between GKO mice of both backgrounds. It is well known that there is a significant difference in susceptibility to acute and chronic infections of T. gondii among inbred mouse strains with different genetic backgrounds. 14,15,17,35,36 It is also understood that genetic polymorphism is responsible for differences in the response to drugs. 37 This mouse model was used successfully to assess treatment effects in a quantitative fashion. The results indicate that study of the kinetics of parasite loads after treatment in the eye may provide additional information on the effect of antimicrobial agents against T. gondii in terms of the evolution of the infection and may represent a reliable starting point for the determination of therapeutic regimens in humans. Using this animal model, several treatments not only currently available agents such as other sulfonamides, pyrimethamine, clindamycin, trimethoprim, azithromycin, and atovaquone, alone and in combination, but also novel, challenging therapies such as chemotherapies combined with cytokines, gene vaccinations, and cell transfer, as well as different strains of T. gondii with varying virulence may be useful in the future. Further studies are needed to determine the optimum treatment type and timing (for example, short-term or long-term and continuous or intermittent therapy) to achieve reduction of the cyst load and thereby reduce the risk of recurrent disease especially important in immunosuppressed patients. Acknowledgments The authors thank Usama S. Belal for invaluable help. References 1. Gagliuso DJ, Teich SA, Friedman AH, Orellana J. Ocular toxoplasmosis in AIDS patients. Trans Am Ophthalmol Soc. 1990;88:63 86; discussion Nicholson DH, Wolchok EB. 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