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Differences between intraocular and serum antibody responses in patients with ocular toxoplasmosis
Differences Between Intraocular and Serum Antibody Responses in Patients With Ocular Toxoplasmosis VINCENT N. A. KLAREN, MS, CLAUDIA E. M. VAN DOORNIK, BS, JENNY V. ONGKOSUWITO, MD, ERIC J. FERON, MD, AND AIZE KIJLSTRA, PHD
● PURPOSE:
To investigate the antigen specificity of the intraocular anti–Toxoplasma gondii antibody response in patients with ocular toxoplasmosis. ● METHODS: Paired ocular fluid and serum samples were collected from 13 patients with active ocular toxoplasmosis. Serum IgM anti–T. gondii antibodies were tested to distinguish recently-acquired from chronic infection. Anti–T. gondii IgG specificity was analyzed by immunoblotting using a crude T. gondii extract. ● RESULTS: Two of the 13 patients tested were IgM positive and considered to have acquired ocular toxoplasmosis. The antibody specificity in ocular fluid and serum of these two patients was similar, whereas in the patients with presumed chronic disease, marked differences could be observed. Most ocular fluid samples contained antibodies that stained a 28-kD antigen more intensely than did antibodies from paired serum samples. Using absorption and elution experiments, we demonstrated that this 28-kD protein was identical to the GRA-2 antigen, which is expressed in Accepted for publication March 17, 1998. From the Department of Ophthalmo-Immunology, The Netherlands Ophthalmic Research Institute, Amsterdam (Dr Klaren, Ms van Doornik, and Drs Ongkosuwito and Kijlstra); The Rotterdam Eye Hospital, Rotterdam (Dr Feron); and the Department of Ophthalmology, University of Amsterdam, Amsterdam (Dr Kijlstra), The Netherlands. This study was funded by the Landelijke Stichting voor Blinden en Slechtzienden, the Stichting Blindenpenning, the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, and the Rotterdamse vereniging Blindenbelangen. Correspondence to Vincent N. A. Klaren, Department of Ophthalmo-Immunology, Netherlands Ophthalmic Research Institute, P.O. Box 12141, 1100 AC Amsterdam, The Netherlands; fax: 1/31-206916521; e-mail: [email protected]
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both the tachyzoite and the bradyzoite stages of the parasite. ● CONCLUSIONS: Our results show that the intraocular T. gondii antibody response of patients with recurrent ocular toxoplasmosis differs from the systemic response. This finding may have implications for our understanding of the immunopathogenesis of ocular toxoplasmosis and could be employed to improve diagnosis of the disease. (Am J Ophthalmol 1998;126:698 –706. © 1998 by Elsevier Science Inc. All rights reserved.)
R
ECURRENT OCULAR TOXOPLASMOSIS IS THE
most frequent cause of posterior uveitis and is an important cause of blindness throughout the world.1 It is considered a late manifestation of a congenital infection.2,3 In some cases, uveitis may also be the result of an acquired infection.4,5 Activation of ‘dormant’ tissue cysts in the neural retina precedes inflammation of the uvea. As a consequence, the retina is irreversibly damaged, which may lead to loss of vision. The process of reactivation of the cysts in the retina is not well understood. It is not known why reactivations in the retina, in contrast to the brain, generally occur in the absence of detectable immunodeficiencies.6 Based on pathologic findings, Frenkel7 suggested that cysts rupture intermittently and give rise to lesions. Spontaneous release of bradyzoites from the cysts has been proposed as a mechanism of maintaining long-lasting immunity during the chronic stage of the infection by boosting the immunoresponse.8 Once escaped from the cysts, the bradyzoites develop into the
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actively proliferating tachyzoite stage, which is capable of infecting adjacent cells. The clinical diagnosis of ocular toxoplasmosis can be confirmed by analysis of intraocular parasitespecific antibody production and is accepted as indirect proof of the presence of the parasite within the eye.9,10 The intraocular antibody response is usually determined using an immunofluorescence technique employing the whole parasite. Since this provides quantitative, but not qualitative, information, we considered it of interest to analyze the antigen specificity of locally produced anti–Toxoplasma gondii IgG, and therefore compared ocular fluid samples with paired serum samples using an immunoblotting technique. In most paired samples, the anti–T. gondii IgG antigen specificity in the eye differed from the antigen specificity in the sera. One of the antigens preferentially recognized by IgG from the ocular samples of patients with ocular toxoplasmosis was identified as GRA-2, a tachyzoite antigen that is also present in the encysted stage of the parasite.11
PATIENTS AND METHODS VITREOUS SAMPLES OR AQUEOUS HUMOR SAMPLES
and paired serum samples of uveitis patients presenting as diagnostic dilemma are routinely sent to our institute by ophthalmologists associated with the Dutch Uveitis Study Group.10 Samples are routinely tested for intraocular antibody production against T. gondii, herpes simplex virus, varicella zoster virus, and cytomegalovirus, and for the presence of DNA by polymerase chain reaction. Remaining samples are stored in an ocular fluid/serum bank at 220 C. As a rule, vitrectomy was performed to clear the vitreous; aqueous humor sampling was performed for diagnostic purposes. Until now, no serious complications have been reported after aqueous humor sampling.12 As described earlier, local antibody production was determined.9 Antitoxoplasma IgG titers were measured briefly with a commercially available immunofluorescence test kit (Toxoplasma suspension OTGY 10/11, fluorescein isothiocyanate-labeled anti-human immunoglobulin OUHT 04/05; Behringwerke AG, Marburg, Germany). Total IgG conVOL. 126, NO. 5
centrations in the samples were determined by radial immunodiffusion.13 Intraocular antibody production was determined by calculation of the Goldmann-Wittmer coefficient,14 which is the quotient of the relative amounts of antitoxoplasma antibodies in the eye and serum and is calculated as follows: antibod y titer ocular fluid antibod y titer serum : total IgG ocular fluid total IgG serum A Goldmann-Wittmer coefficient of greater than 3 is considered as evidence for T. gondii–specific antibody production in the eye with a test specificity of 100% and a sensitivity of 74%,15 and is sufficient for diagnosis of ocular toxoplasmosis. For this study, patient samples from 1990 to 1995 were selected from our database according to the following criteria: Goldmann-Wittmer coefficient of greater than 3 for T. gondii; Goldmann-Wittmer coefficient of less than 3 for other infectious microorganisms; and anti–T. gondii antibody titers in serum and ocular fluid of at least 1/64. From 104 patients with ocular toxoplasmosis, 54 met both criteria. Sufficient sample material was available for analysis of the 13 cases presented in this study. Sera were screened for T. gondii–specific IgM antibodies with a capture ELISA method (Toxoplasma IgM ELISA System Capture Method Kit; Alfa Biotech, Rome, Italy). Control samples (n 5 4) were selected from our diagnostic database if posterior uveitis was caused by an infectious agent other than T. gondii or one of noninfectious origin, and if anti–T. gondii antibody titers in serum and ocular fluid measured at least 1/64. Two control patients had acute retinal necrosis caused by herpes simplex virus and varicella-zoster virus, respectively. One patient had uveitis due to a Candida albicans infection, and one patient had Behc¸et disease. Clinical information about the patients was obtained from the records of the hospitals and from written and telephone communications with the referring physicians. Informed consent was obtained from all patients. A crude T. gondii extract was prepared according to a method that has been previously described16 and is summarized below. Briefly, tachyzoites from the T. gondii RH strain were obtained from the
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bodies (Nordic Immunological Laboratories, Tilburg, The Netherlands) at a dilution of 1/200 in phosphate buffered saline 0.1% Tween-20 with 1% casein for 1 hour. After they were washed four times, the strips were soaked in a solution of 1 mg/ml of diaminobenzidine 0.1% hydrogen peroxide in phosphate buffered saline. The color development was stopped by washing with phosphate buffered saline after 5 to 10 minutes’ incubation. Controls included incubation of blots with the conjugate alone and with serum and ocular fluid samples not containing detectable anti–T. gondii antibodies with the immunofluorescent assay (data not shown). Immunoblots were scanned and analyzed using ImageMaster software (Pharmacia; Upsala, Sweden). Individual bands were quantitated and expressed as a percent of total staining within the lane ranging between 25 and 80 kD. To identify the 28-kD band as GRA-2, a DNA plasmid encoding GRA-2 as a fusion protein with glutathione Stransferase (rGRA-2) (provided by S. F. Parmley, Palo Alto, California) was expressed in E. coli Jm101 and purified by affinity to glutathione agarose.17 Circular pieces of nitrocellulose membrane (diameter, 5 mm) were spotted with 50-mg rGRA-2 to absorb anti-GRA-2 antibodies from the patient samples. After blocking with phosphate buffered saline 0.1% Tween-20 with 1% casein, the membranes were incubated overnight with serum or ocular fluid from Patient 10. Serum was diluted 1/16 and ocular fluid was diluted 1/32 in 100 ml phosphate buffered saline 0.1% Tween-20 with 1% casein. Dilution of the samples was based on the anti–T. gondii antibody titer, as measured with the immunofluorescent assay, to obtain comparable specific antibody concentrations. The membranes were washed three times with phosphate buffered saline Tween-20 and rinsed with 66 ml elution buffer (50 mM sodium chloride, 50 mM glycine hydrochloride pH 2.3, 0.5% Tween-20 with 1% casein) to elute bound antibodies. The eluates were neutralized to pH 7.4 by addition of 25 mM sodium dihydrogen phosphate, 0.5% Tween-20 with 1.4% casein to a final volume of 250 ml and used to stain Western blot strips as described above. Serum sample (1/800), ocular fluid sample (1/ 1600) from Patient 10, and the eluates prepared as
peritoneal cavity of mice 72 hours after intraperitoneal inoculation. Host cells were disrupted by forced passage through a 27.5-gauge needle and tachyzoites were washed three times in phosphate buffered saline and were pelleted by centrifugation (750 3 g for 10 minutes). After three freeze-thaw cycles, the parasites were subjected to pulsed sonication (5 3 15 seconds 30 kHz microprobe, Soniprep 150; Making Science Effective, Loughborough, United Kingdom). The lysate was centrifuged at 15,600 3 g for 30 minutes, and the supernatant was concentrated using an ultrafiltration membrane with a cutoff at 10 kDa (type PLGC membrane; Millipore SA, Molsheim, France) and stored at 270 degrees. Polyacrylamide gel electrophoresis was performed with 2 ml of lysate in a sample buffer at a final concentration of 2% sodium dodecyl sulfate, 63 mM Tris-HCl, 10% glycerol, 0.01% bromophenol blue using 10% to 15% gradient slab gels with a discontinuous sodium dodecyl sulfate buffer system (PhastSystem; Pharmacia, Upsala, Sweden). A standard broad range mix of proteins (Bio-Rad Laboratories; Hercules, California) was used as marker for the determination of the approximate molecular weights of the bands. Protein was transferred to polyvinylidene fluoride membranes (Immobilon-P PVDF, 0.45-mm pore size; Millipore, Bedford, Massachusetts) with a semi-dry transfer unit (PhastTransfer; Pharmacia, Upsala, Sweden) for 2 hours at 7 volts. Transfer buffer was 25 mM Tris-HCl, 192 mM glycine, and 20% volume per volume methanol (pH 8.3). To saturate unused protein binding sites, the membranes were soaked in a solution of phosphate buffered saline 0.1% Tween-20 containing 1% casein for 1 hour. The membrane was cut into strips of 4 mm in width. For immunoblotting, sample dilutions were normalized according to their antitoxoplasma antibody titer, as measured with the immunofluorescent assay. For example, samples with an immunofluorescent titer of 1/64 were diluted 1/100 (1/128, 1/200, 1/256, 1/400, and so on). The strips were incubated overnight with the diluted sera and ocular fluid samples (400-ml phosphate buffered saline 0.1% Tween-20 with 1% casein). The strips were then washed four times with phosphate buffered saline 0.1% Tween-20 and incubated with horseradish peroxidase-conjugated goat-anti-human-IgG anti700
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TABLE. Patient Data Case No., Age (yrs), Sex
Toxoplasma IgM* (serum)
Toxoplasma Serum
IgG Titer† Ocular Fluid
Goldmann-Wittmer Coefficient
Episode Number
1, 25, M
2
1/512
1/64 (A)
23
.1
2, 74, F
1
1/128
1/512 (V)
10
1
3, 67, M 4, 43, M
2 2
1/128 1/1,024
1/64 (V) 1/128 (A)
5, 6, 7, 8, 9,
2 2 1 2 2
1/1,024 1/1,024 1/2,048 1/256 1/128
10, 41, M
2
11, 61, F 12, 37, F 13, 75, F
28, 27, 50, 52, 19,
F M M F M
4.4 3.2
.2 11
1/1,024 (A) 1/64 (A) 1/1,024 (A) 1/256 (A) 1/256 (V)
3.8 4.8 38 25 27
.2 .2 3 .2 .2
1/1,024
1/2,048 (A)
7.8
.2
2 2
1/512 1/128
1/128 (V) 1/512 (V)
6.0 110
.3 .3
2
1/1,024
1/512 (V)
11
7
Ophthalmic Signs at Presentation
Panuveitis, focal CHR, dense vitreous opacities, scar Necrotizing retinopathy, no old scars Uveitis, two scars Vitreous opacities, scars, focal CHR Focal CHR Focal CHR, vasculitis, scar Focal CHR, anterior uveitis Scars with border activity Fibrotic membrane over macula with scar Vitritis, total retinal detachment Focal CHR, two scars Focal CHR, vitreous opacities Focal CHR, vitreous opacities
A 5 aqueous humor; CHR 5 chorioretinitis; V 5 vitreous fluid. *Tested with IgM capture ELISA; †Expressed as dilution factor.
described above were incubated overnight with Western blot strips containing T. gondii lysate antigens. Bound IgG was developed as described above. One Western blot strip was stained for GRA-2 with a monoclonal anti-GRA-2 antibody (provided by M. F. Cesbron, Lille, France), and developed as described above using goat-antimouse-IgG (Nordic, Tilburg, The Netherlands). The above described procedure was also performed with solid phase bound glutathione S-transferase, the fusion part of the recombinant GRA-2, and serum of Patient 10.
RESULTS THE OCULAR TOXOPLASMOSIS PATIENTS USED FOR
this study all had the clinical picture of the disease in combination with proven intraocular antibody synthesis against the parasite (Table). The Goldmann-Wittmer coefficients for IgG of the patient samples ranged between 3.2 and 110. Parasite-specific IgM was detected in the sera of two patients. Patient 2 was immunocompromised VOL. 126, NO. 5
and had chronic lymphatic leukemia. No old retinal scars were observed. Patient 7 had three attacks of panuveitis in the same eye during a 22-month period, with high IgG titers and persistent IgM antibodies. This was tested on three separate occasions. After this period, the serum became IgM negative. These two patients were considered to have acutely acquired ocular toxoplasmosis. All of the other patients had two or more recurrences, and their sera were IgM negative. These patients were considered to have recurrent (chronic) ocular toxoplasmosis. The immunoblots performed using sera and ocular fluids from patients with ocular toxoplasmosis showed a diverse banding pattern (Figure 1). Differences in banding patterns are visible both within and between the paired samples. The bands, which are clearly recognized by most of the samples, correspond to an approximate molecular weight of 28, 30, 35, 38, 43 (doublets), 50, 58 (doublets), 63, and 73 kD. The bands outside the range between 25 and 80 kD were not analyzed. The ocular fluid samples of 10 of the 13 patients stained a band of approximately 28 kD more intense
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FIGURE 1. Immunoblot analysis of a T. gondii crude extract recognized by IgG from serum samples (S) and ocular fluid samples (OF) of 13 patients with ocular toxoplasmosis (the lane numbers correlate with patient numbers given in the Table). Arrows on the right point to the 28-, 30-, and 43-kD bands. Numbers on the left represent molecular weight markers in thousands.
the 28-kD band of the 13 patients was 3.6 (SD 6 3.9). This indicates an overall stronger anti-28-kD response in the eyes of patients with ocular toxoplasmosis. The mean ocular fluid to serum ratio of the 30-kD band was 0.85 (SD 6 0.45), indicating a slightly weaker intraocular anti-30-kD response compared to the response in the serum. The difference in response between the 30- and the 28-kD
than the paired serum samples did. Figure 2 shows a plot of densitometer scans of the lanes of Patient 10 on the immunoblot (within the range of 25 and 80 kD). Staining of the 28-kD band (relative front 5 0.87) and the 30-kD band (relative front 5 0.8) was quantitated as a percentage of total staining and used to calculate an ocular fluid to serum ratio (Figure 3). The mean ocular fluid to serum ratio of 702
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FIGURE 2. Densitometer scans of the lanes of Patient 10 on the immunoblot shown in Figure 1 within the range of 25 to 80 kD. The solid line represents serum; the dotted line represents ocular fluid. The plots of each lane are determined in the x-axis by relative front values (relative front 5 distance of protein migration per length of the lane) and in the y-axis by optical density values. The 30-kD band corresponds to the peaks at relative front 5 0.80 and the 28-kD band corresponds to the peaks at relative front 5 0.87.
bands was found to be statistically significant (P 5 .0175) by using the Wilcoxon signed rank test. A band of 38 kD was stained more intensely by eight of 13 sera compared with staining by paired ocular fluid samples. Bands with approximate molecular weights of 58 and 43 kD were more intensely stained by ocular fluid samples in nine of 13 sample pairs. The samples of the two IgM positive patients did not disclose differences in staining of the above mentioned bands. The control samples from four patients with uveitis from causes other than T. gondii without intraocular anti–T. gondii antibody production but with a sufficiently high titer in ocular fluids all stained the 30-kD band. The samples of Patient 1 also stained a band of apparent molecular weight of 38 kD. No differences in staining patterns between the ocular fluid and the paired serum samples were observed (Figure 4). The ocular fluid to serum ratio of the 30-kD band was 0.90 (SD 6 0.67), and the VOL. 126, NO. 5
ratio of the 28-kD band was 0.94 (SD 6 0.57). No bands were disclosed after immunoblotting with serum from patients without an anti–T. gondii antibody titer or the conjugated antibody alone (data not shown). To investigate whether the reactivity of IgG from ocular fluids against the 28-kD antigen in the crude T. gondii extract was directed to the so-called GRA-2 (granule antigen 2),18 specific IgG from ocular fluid and serum of Patient 10 was absorbed with solid phase bound recombinant GRA-2. After washing and elution (see Methods), eluted IgG was tested with immunoblotting using the T. gondii extract. The eluted serum-IgG only weakly stained at the 28-kD position and had some background staining of the 30-kD band (Figure 5, lane 2), whereas the eluate from the ocular fluid showed evident staining of the 28-kD band (Figure 5, lane 4). The presence of GRA-2 in our T. gondii antigen extract was verified using a monoclonal anti-GRA-2
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FIGURE 3. Graph of relative intensities of the 30- and 28-kD bands of the T. gondii immunoblots as seen in Figure 1. Ratios were calculated of relative intensities of bands per lane of ocular fluid and serum. Ratios higher than 1 indicate a stronger band stained by ocular fluid compared with bands stained by serum.
antibody, which clearly disclosed a strong band at the 28-kD position (Figure 5, lane 5). Solid phase bound glutathione S-transferase, the fusion part of the recombinant GRA-2, was used as a control. After incubation with serum (ocular fluid was no longer available), the eluate did not stain any bands on immunoblot (data not shown). These findings make it very likely that the 28-kD band is identical to GRA-2 and furthermore confirm the observation that ocular fluid of this patient contains relatively more anti-GRA-2 antibodies than the serum does. The same experiment was performed with samples from Patient 13 showing identical results (data not shown).
FIGURE 4. Patients with posterior uveitis caused by an infectious agent other than T. gondii but with anti–T. gondii antibody titers both in the serum and ocular fluid. The infective agents of the inflammation of the eyes are represented at the bottom as follows: 1 5 herpes simplex virus; 2 5 Candida albicans; 3 5 varicella-zoster virus; 4 5 presumed autoimmune uveitis. Numbers on the left represent molecular weight markers in thousands.
patients with ocular toxoplasmosis in this study leads to the supposition that some T. gondii antigens may be more abundant within the eyes of these patients. The paired samples from the two IgM positive patients (with presumed acutely acquired infection) do not disclose these differences in recognition of antigens. These observations suggest a relation between the intraocular antibody response against the 28-, 43-, and 58-kD antigens and a recurrent, chronic infection with T. gondii. Recurrent ocular toxoplasmosis is thought to be due to reactivating cysts within the retina, and it is conceivable that the intraocular immunoresponse is partly directed against antigens from these cysts. Darcy and associates11 showed that a 28.5-kD antigen from a tachyzoite extract was recognized by mouse sera obtained from animals immunized with a
DISCUSSION THE FACT THAT OCULAR TOXOPLASMOSIS IS ASSOCI-
ated with a strong intraocular antibody response has been known for several decades19 and is routinely used for diagnostic purposes. Intraocular toxoplasma antibody testing involves an immunofluorescence technique using intact parasites and thus provides information about the quantity but not the specificity of the parasite antibody response in the eye. The more pronounced recognition of a 28-kD antigen (and to a lesser extent a 43-kD and a 58-kD antigen) by anti–T. gondii IgG from the eyes of most 704
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FIGURE 5. Results of elution experiment with serum and ocular fluid of Patient 10 are shown. After incubation of serum and ocular fluid with solid phase bound rGRA-2 and extensive washing, bound antibodies were eluted in an acid buffer, neutralized, and used for immunoblot analysis of a T. gondii crude extract. Elution samples are 50 times more concentrated. On the bottom, the number 1 represents serum and 3 represents ocular fluid; for eluates of solid phase bound rGRA-2, 2 represents serum and 4 represents ocular fluid; 5 represents monoclonal anti–GRA-2 antibody. Numbers on the left represent molecular weight markers in thousands.
bradyzoite extract. This antigen, later identified as GRA-2,20 is identical to the antigen to which a majority of the ocular toxoplasmosis patients in this study shows a relatively strong intraocular antibody response. This statement is based on absorption and elution experiments with recombinant GRA-2 and the demonstration of the presence of GRA-2 in our crude T. gondii extract using a monoclonal antiGRA-2 antibody. Darcy and associates11 also identified a 43-kD bradyzoite antigen that was recognized by chronic VOL. 126, NO. 5
phase human sera. Whether this antigen, later designated as SAG-3, is similar to the 43-kD band reactivity observed in the study presented here has yet to be established. Of interest is that both the GRA-2 and SAG-3 antigens are expressed by both the tachyzoite as well as the bradyzoite stages of the parasite. This may support the hypothesis that the immunoresponse of patients with chronic recurring ocular toxoplasmosis is driven by bradyzoite antigens leaking from the cysts. Assuming that the initial systemic infection leads to the priming of the immunoresponse against the tachyzoite stage of the parasite, one would assume that a subsequent intraocular immunoresponse driven by antigens leaking from the cysts is possibly directed against antigens shared between bradyzoites and tachyzoites. In the acquired form of the disease, systemic humoral immunity is mainly directed to tachyzoite antigens whereby selection of the intraocular response, when compared with the systemic response, is not yet likely since cyst formation will probably occur later in the disease. Our data showing a similar specificity of the ocular vs the systemic response in two patients with presumed acquired ocular toxoplasmosis would favor this speculative hypothesis. Definitive proof of this hypothesis would involve a comparison of the intraocular immunoresponse with a variety of bradyzoite and tachyzoite antigens. The major surface antigen, SAG-1, with a molecular weight of 30 kD, is considered to be the most abundant surface protein of T. gondii.21 Almost all individuals develop a high serum antibody titer against SAG-1 after exposure to T. gondii; this is one of the earliest, most consistent signs of toxoplasma infection.18,22–24 SAG-1 is the principle antigen recognized by immune antisera from both chronic and acutely-infected individuals.25 The consistent staining of a 30-kD band in our experiments most likely reflects reactivity against SAG-1. Titers obtained with the indirect immunofluorescence assay are probably to a large extent based on the recognition of this surface antigen. Due to the fact that we normalized serum and ocular fluid samples with respect to their immunofluorescent assay anti–T. gondii titers, the 30-kD band on the immunoblots of most paired samples stained with similar intensities.
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10. Kijlstra A, Rothova A, Baarsma GS, et al. Computer registration of uveitis patients. Doc Ophthalmol 1987; 67:139 –143. 11. Darcy F, Charif H, Caron H, et al. Identification and biochemical characterization of antigens of tachyzoites and bradyzoites of Toxoplasma gondii with cross-reactive epitopes. Parasitol Res 1990;76:473– 478. 12. Van der Lelij A, Rothova A. Diagnostic anterior chamber paracentesis in uveitis: a safe procedure? Br J Ophthalmol 1997;81:976 –979. 13. Mancini G, Carbonara AO, Heremans JF. Immunochemical quantitation of antisera by single radial immunodiffusion. Immunohistochemistry 1965;2:235–256. 14. Goldmann H, Wittmer R. Antikorper im kammerwasser. Ophthalmologica 1954;127:323–230. 15. Luyendijk L, de Boer JH, Rothova A, et al. Sensitivity and specificity of anti-viral antibody determination in the aqueous or vitreous of uveitis patients. In: Dernouchamps JP, Verougstreate I, Caspers-Velu I, Tassignon MJ, editors. Recent advances in uveitis. Amsterdam: Kugler, 1993:265– 267. 16. Bessieres MH, Le Breton S, Seguela JP. Analysis by immunoblotting of Toxoplasma gondii exo-antigens and comparison with somatic antigens. Parasitol Res 1992;78: 222–228. 17. Parmley SF, Sgarlato GD, Mark J, Prince JB, Remington JS. Expression, characterization, and serologic reactivity of recombinant surface antigen P22 of Toxoplasma gondii. J Clin Microbiol 1992;30:1127–1133. 18. Potasman I, Araujo FG, Desmonts G, Remington JS. Analysis of Toxoplasma gondii antigens recognized by human sera obtained before and after acute infection. J Infect Dis 1986;154:650 – 657. 19. Desmonts G. Definitive serological diagnosis of ocular toxoplasmosis. Arch Ophthalmol 1966;76:839 – 851. 20. Parmley SF, Sgarlato GD, Remington JS. Genomic and corrected cDNA sequence of the P28 gene from Toxoplasma gondii. Mol Biochem Parasitol 1993;57:161–165. 21. Kasper LH, Crabb JH, Pfefferkorn ER. Purification of a major membrane protein of Toxoplasma gondii by immunoabsorption with a monoclonal antibody. J Immunol 1983; 130:2407–2412. 22. Remington JS, Araujo FG, Desmonts G. Recognition of different Toxoplasma antigens by IgM and IgG antibodies in mothers and their congenitally infected newborns. J Infect Dis 1985;152:1020 –1024. 23. Santoro F, Afchain D, Pierce R, Cesbron JY, Ovlaque G, Capron A. Serodiagnosis of toxoplasma infection using a purified parasite protein (P30). Clin Exp Immunol 1985; 62:262–269. 24. Sharma SD, Mullenax J, Araujo FG, Erlich HA, Remington JS. Western Blot analysis of the antigens of Toxoplasma gondii recognized by human IgM and IgG antibodies. J Immunol 1983;131:977–983. 25. Decoster A, Darcy F, Caron A, Capron A. IgA antibodies against P30 as markers of congenital and acute toxoplasmosis. Lancet 1988;2:1104 –1107.
Since all patients in our study were reported as a diagnostic dilemma and the number of patients was small, our results may only be reflective of this selected group. Future studies are required to draw more general conclusions about the intraocular antibody response and may improve our knowledge concerning the pathogenesis of ocular toxoplasmosis, which can lead to further refinement of the laboratory diagnosis of this disease. ACKNOWLEDGMENT
We wish to thank members of the Dutch Uveitis Study Group for their help concerning patient samples and clinical information. We thank Dr T. van Gool from the Department of Microbiology, University of Amsterdam, for providing us with the T. gondii tachyzoites.
REFERENCES 1. Nussenblatt RB, Palestine AG. Ocular toxoplasmosis. In: Marshall DK, editor. Uveitis: fundamentals and clinical practice. Chicago: Yearbook Medical Publishers, 1989: 336 –354. 2. Loewer-Sieger DH, Rothova A, Koppe JG, et al. Congenital toxoplasmosis: a prospective study based on 1821 pregnant women. In: Saari KM, editor. Uveitis update. Amsterdam: Elsevier, 1984:203–207. 3. Frenkel JK. Pathophysiology of toxoplasmosis. Parasitol Today 1988;4:273–278. 4. Glasner PD, Silveira C, Kruszon-Moran D, et al. An unusually high prevalence of ocular toxoplasmosis in southern Brazil. Am J Ophthalmol 1992;114:136 –144. 5. Ronday MJ, Luyendijk L, Baarsma GS, Bollemeijer JG, Van der Lelij A, Rothova A. Presumed acquired ocular toxoplasmosis. Arch Ophthalmol 1995;113:1524 –1529. 6. Ambroise-Thomas P, Pelloux H. Toxoplasmosis— congenital and in immunocompromised patients: a parallel. Parasitol Today 1993;9:61– 63. 7. Frenkel JK. Host, strain and treatment variation as factors in the pathogenesis of toxoplasmosis. Am J Trop Med Hyg 1953;2:390 – 415. 8. Gazzinelli RT, Denkers EY, Sher A. Host resistance to Toxoplasma gondii: model for studying the selective induction of cell-mediated immunity by intracellular parasites. Infect Agents Dis 1993;2:139 –149. 9. Kijlstra A, Luyendijk L, Baarsma GS, et al. Aqueous humor analysis as a diagnostic tool in toxoplasma uveitis. Int Ophthalmol 1989;13:383–386.
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● PURPOSE:
To investigate the antigen specificity of the intraocular anti–Toxoplasma gondii antibody response in patients with ocular toxoplasmosis. ● METHODS: Paired ocular fluid and serum samples were collected from 13 patients with active ocular toxoplasmosis. Serum IgM anti–T. gondii antibodies were tested to distinguish recently-acquired from chronic infection. Anti–T. gondii IgG specificity was analyzed by immunoblotting using a crude T. gondii extract. ● RESULTS: Two of the 13 patients tested were IgM positive and considered to have acquired ocular toxoplasmosis. The antibody specificity in ocular fluid and serum of these two patients was similar, whereas in the patients with presumed chronic disease, marked differences could be observed. Most ocular fluid samples contained antibodies that stained a 28-kD antigen more intensely than did antibodies from paired serum samples. Using absorption and elution experiments, we demonstrated that this 28-kD protein was identical to the GRA-2 antigen, which is expressed in Accepted for publication March 17, 1998. From the Department of Ophthalmo-Immunology, The Netherlands Ophthalmic Research Institute, Amsterdam (Dr Klaren, Ms van Doornik, and Drs Ongkosuwito and Kijlstra); The Rotterdam Eye Hospital, Rotterdam (Dr Feron); and the Department of Ophthalmology, University of Amsterdam, Amsterdam (Dr Kijlstra), The Netherlands. This study was funded by the Landelijke Stichting voor Blinden en Slechtzienden, the Stichting Blindenpenning, the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, and the Rotterdamse vereniging Blindenbelangen. Correspondence to Vincent N. A. Klaren, Department of Ophthalmo-Immunology, Netherlands Ophthalmic Research Institute, P.O. Box 12141, 1100 AC Amsterdam, The Netherlands; fax: 1/31-206916521; e-mail: [email protected]
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both the tachyzoite and the bradyzoite stages of the parasite. ● CONCLUSIONS: Our results show that the intraocular T. gondii antibody response of patients with recurrent ocular toxoplasmosis differs from the systemic response. This finding may have implications for our understanding of the immunopathogenesis of ocular toxoplasmosis and could be employed to improve diagnosis of the disease. (Am J Ophthalmol 1998;126:698 –706. © 1998 by Elsevier Science Inc. All rights reserved.)
R
ECURRENT OCULAR TOXOPLASMOSIS IS THE
most frequent cause of posterior uveitis and is an important cause of blindness throughout the world.1 It is considered a late manifestation of a congenital infection.2,3 In some cases, uveitis may also be the result of an acquired infection.4,5 Activation of ‘dormant’ tissue cysts in the neural retina precedes inflammation of the uvea. As a consequence, the retina is irreversibly damaged, which may lead to loss of vision. The process of reactivation of the cysts in the retina is not well understood. It is not known why reactivations in the retina, in contrast to the brain, generally occur in the absence of detectable immunodeficiencies.6 Based on pathologic findings, Frenkel7 suggested that cysts rupture intermittently and give rise to lesions. Spontaneous release of bradyzoites from the cysts has been proposed as a mechanism of maintaining long-lasting immunity during the chronic stage of the infection by boosting the immunoresponse.8 Once escaped from the cysts, the bradyzoites develop into the
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actively proliferating tachyzoite stage, which is capable of infecting adjacent cells. The clinical diagnosis of ocular toxoplasmosis can be confirmed by analysis of intraocular parasitespecific antibody production and is accepted as indirect proof of the presence of the parasite within the eye.9,10 The intraocular antibody response is usually determined using an immunofluorescence technique employing the whole parasite. Since this provides quantitative, but not qualitative, information, we considered it of interest to analyze the antigen specificity of locally produced anti–Toxoplasma gondii IgG, and therefore compared ocular fluid samples with paired serum samples using an immunoblotting technique. In most paired samples, the anti–T. gondii IgG antigen specificity in the eye differed from the antigen specificity in the sera. One of the antigens preferentially recognized by IgG from the ocular samples of patients with ocular toxoplasmosis was identified as GRA-2, a tachyzoite antigen that is also present in the encysted stage of the parasite.11
PATIENTS AND METHODS VITREOUS SAMPLES OR AQUEOUS HUMOR SAMPLES
and paired serum samples of uveitis patients presenting as diagnostic dilemma are routinely sent to our institute by ophthalmologists associated with the Dutch Uveitis Study Group.10 Samples are routinely tested for intraocular antibody production against T. gondii, herpes simplex virus, varicella zoster virus, and cytomegalovirus, and for the presence of DNA by polymerase chain reaction. Remaining samples are stored in an ocular fluid/serum bank at 220 C. As a rule, vitrectomy was performed to clear the vitreous; aqueous humor sampling was performed for diagnostic purposes. Until now, no serious complications have been reported after aqueous humor sampling.12 As described earlier, local antibody production was determined.9 Antitoxoplasma IgG titers were measured briefly with a commercially available immunofluorescence test kit (Toxoplasma suspension OTGY 10/11, fluorescein isothiocyanate-labeled anti-human immunoglobulin OUHT 04/05; Behringwerke AG, Marburg, Germany). Total IgG conVOL. 126, NO. 5
centrations in the samples were determined by radial immunodiffusion.13 Intraocular antibody production was determined by calculation of the Goldmann-Wittmer coefficient,14 which is the quotient of the relative amounts of antitoxoplasma antibodies in the eye and serum and is calculated as follows: antibod y titer ocular fluid antibod y titer serum : total IgG ocular fluid total IgG serum A Goldmann-Wittmer coefficient of greater than 3 is considered as evidence for T. gondii–specific antibody production in the eye with a test specificity of 100% and a sensitivity of 74%,15 and is sufficient for diagnosis of ocular toxoplasmosis. For this study, patient samples from 1990 to 1995 were selected from our database according to the following criteria: Goldmann-Wittmer coefficient of greater than 3 for T. gondii; Goldmann-Wittmer coefficient of less than 3 for other infectious microorganisms; and anti–T. gondii antibody titers in serum and ocular fluid of at least 1/64. From 104 patients with ocular toxoplasmosis, 54 met both criteria. Sufficient sample material was available for analysis of the 13 cases presented in this study. Sera were screened for T. gondii–specific IgM antibodies with a capture ELISA method (Toxoplasma IgM ELISA System Capture Method Kit; Alfa Biotech, Rome, Italy). Control samples (n 5 4) were selected from our diagnostic database if posterior uveitis was caused by an infectious agent other than T. gondii or one of noninfectious origin, and if anti–T. gondii antibody titers in serum and ocular fluid measured at least 1/64. Two control patients had acute retinal necrosis caused by herpes simplex virus and varicella-zoster virus, respectively. One patient had uveitis due to a Candida albicans infection, and one patient had Behc¸et disease. Clinical information about the patients was obtained from the records of the hospitals and from written and telephone communications with the referring physicians. Informed consent was obtained from all patients. A crude T. gondii extract was prepared according to a method that has been previously described16 and is summarized below. Briefly, tachyzoites from the T. gondii RH strain were obtained from the
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bodies (Nordic Immunological Laboratories, Tilburg, The Netherlands) at a dilution of 1/200 in phosphate buffered saline 0.1% Tween-20 with 1% casein for 1 hour. After they were washed four times, the strips were soaked in a solution of 1 mg/ml of diaminobenzidine 0.1% hydrogen peroxide in phosphate buffered saline. The color development was stopped by washing with phosphate buffered saline after 5 to 10 minutes’ incubation. Controls included incubation of blots with the conjugate alone and with serum and ocular fluid samples not containing detectable anti–T. gondii antibodies with the immunofluorescent assay (data not shown). Immunoblots were scanned and analyzed using ImageMaster software (Pharmacia; Upsala, Sweden). Individual bands were quantitated and expressed as a percent of total staining within the lane ranging between 25 and 80 kD. To identify the 28-kD band as GRA-2, a DNA plasmid encoding GRA-2 as a fusion protein with glutathione Stransferase (rGRA-2) (provided by S. F. Parmley, Palo Alto, California) was expressed in E. coli Jm101 and purified by affinity to glutathione agarose.17 Circular pieces of nitrocellulose membrane (diameter, 5 mm) were spotted with 50-mg rGRA-2 to absorb anti-GRA-2 antibodies from the patient samples. After blocking with phosphate buffered saline 0.1% Tween-20 with 1% casein, the membranes were incubated overnight with serum or ocular fluid from Patient 10. Serum was diluted 1/16 and ocular fluid was diluted 1/32 in 100 ml phosphate buffered saline 0.1% Tween-20 with 1% casein. Dilution of the samples was based on the anti–T. gondii antibody titer, as measured with the immunofluorescent assay, to obtain comparable specific antibody concentrations. The membranes were washed three times with phosphate buffered saline Tween-20 and rinsed with 66 ml elution buffer (50 mM sodium chloride, 50 mM glycine hydrochloride pH 2.3, 0.5% Tween-20 with 1% casein) to elute bound antibodies. The eluates were neutralized to pH 7.4 by addition of 25 mM sodium dihydrogen phosphate, 0.5% Tween-20 with 1.4% casein to a final volume of 250 ml and used to stain Western blot strips as described above. Serum sample (1/800), ocular fluid sample (1/ 1600) from Patient 10, and the eluates prepared as
peritoneal cavity of mice 72 hours after intraperitoneal inoculation. Host cells were disrupted by forced passage through a 27.5-gauge needle and tachyzoites were washed three times in phosphate buffered saline and were pelleted by centrifugation (750 3 g for 10 minutes). After three freeze-thaw cycles, the parasites were subjected to pulsed sonication (5 3 15 seconds 30 kHz microprobe, Soniprep 150; Making Science Effective, Loughborough, United Kingdom). The lysate was centrifuged at 15,600 3 g for 30 minutes, and the supernatant was concentrated using an ultrafiltration membrane with a cutoff at 10 kDa (type PLGC membrane; Millipore SA, Molsheim, France) and stored at 270 degrees. Polyacrylamide gel electrophoresis was performed with 2 ml of lysate in a sample buffer at a final concentration of 2% sodium dodecyl sulfate, 63 mM Tris-HCl, 10% glycerol, 0.01% bromophenol blue using 10% to 15% gradient slab gels with a discontinuous sodium dodecyl sulfate buffer system (PhastSystem; Pharmacia, Upsala, Sweden). A standard broad range mix of proteins (Bio-Rad Laboratories; Hercules, California) was used as marker for the determination of the approximate molecular weights of the bands. Protein was transferred to polyvinylidene fluoride membranes (Immobilon-P PVDF, 0.45-mm pore size; Millipore, Bedford, Massachusetts) with a semi-dry transfer unit (PhastTransfer; Pharmacia, Upsala, Sweden) for 2 hours at 7 volts. Transfer buffer was 25 mM Tris-HCl, 192 mM glycine, and 20% volume per volume methanol (pH 8.3). To saturate unused protein binding sites, the membranes were soaked in a solution of phosphate buffered saline 0.1% Tween-20 containing 1% casein for 1 hour. The membrane was cut into strips of 4 mm in width. For immunoblotting, sample dilutions were normalized according to their antitoxoplasma antibody titer, as measured with the immunofluorescent assay. For example, samples with an immunofluorescent titer of 1/64 were diluted 1/100 (1/128, 1/200, 1/256, 1/400, and so on). The strips were incubated overnight with the diluted sera and ocular fluid samples (400-ml phosphate buffered saline 0.1% Tween-20 with 1% casein). The strips were then washed four times with phosphate buffered saline 0.1% Tween-20 and incubated with horseradish peroxidase-conjugated goat-anti-human-IgG anti700
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TABLE. Patient Data Case No., Age (yrs), Sex
Toxoplasma IgM* (serum)
Toxoplasma Serum
IgG Titer† Ocular Fluid
Goldmann-Wittmer Coefficient
Episode Number
1, 25, M
2
1/512
1/64 (A)
23
.1
2, 74, F
1
1/128
1/512 (V)
10
1
3, 67, M 4, 43, M
2 2
1/128 1/1,024
1/64 (V) 1/128 (A)
5, 6, 7, 8, 9,
2 2 1 2 2
1/1,024 1/1,024 1/2,048 1/256 1/128
10, 41, M
2
11, 61, F 12, 37, F 13, 75, F
28, 27, 50, 52, 19,
F M M F M
4.4 3.2
.2 11
1/1,024 (A) 1/64 (A) 1/1,024 (A) 1/256 (A) 1/256 (V)
3.8 4.8 38 25 27
.2 .2 3 .2 .2
1/1,024
1/2,048 (A)
7.8
.2
2 2
1/512 1/128
1/128 (V) 1/512 (V)
6.0 110
.3 .3
2
1/1,024
1/512 (V)
11
7
Ophthalmic Signs at Presentation
Panuveitis, focal CHR, dense vitreous opacities, scar Necrotizing retinopathy, no old scars Uveitis, two scars Vitreous opacities, scars, focal CHR Focal CHR Focal CHR, vasculitis, scar Focal CHR, anterior uveitis Scars with border activity Fibrotic membrane over macula with scar Vitritis, total retinal detachment Focal CHR, two scars Focal CHR, vitreous opacities Focal CHR, vitreous opacities
A 5 aqueous humor; CHR 5 chorioretinitis; V 5 vitreous fluid. *Tested with IgM capture ELISA; †Expressed as dilution factor.
described above were incubated overnight with Western blot strips containing T. gondii lysate antigens. Bound IgG was developed as described above. One Western blot strip was stained for GRA-2 with a monoclonal anti-GRA-2 antibody (provided by M. F. Cesbron, Lille, France), and developed as described above using goat-antimouse-IgG (Nordic, Tilburg, The Netherlands). The above described procedure was also performed with solid phase bound glutathione S-transferase, the fusion part of the recombinant GRA-2, and serum of Patient 10.
RESULTS THE OCULAR TOXOPLASMOSIS PATIENTS USED FOR
this study all had the clinical picture of the disease in combination with proven intraocular antibody synthesis against the parasite (Table). The Goldmann-Wittmer coefficients for IgG of the patient samples ranged between 3.2 and 110. Parasite-specific IgM was detected in the sera of two patients. Patient 2 was immunocompromised VOL. 126, NO. 5
and had chronic lymphatic leukemia. No old retinal scars were observed. Patient 7 had three attacks of panuveitis in the same eye during a 22-month period, with high IgG titers and persistent IgM antibodies. This was tested on three separate occasions. After this period, the serum became IgM negative. These two patients were considered to have acutely acquired ocular toxoplasmosis. All of the other patients had two or more recurrences, and their sera were IgM negative. These patients were considered to have recurrent (chronic) ocular toxoplasmosis. The immunoblots performed using sera and ocular fluids from patients with ocular toxoplasmosis showed a diverse banding pattern (Figure 1). Differences in banding patterns are visible both within and between the paired samples. The bands, which are clearly recognized by most of the samples, correspond to an approximate molecular weight of 28, 30, 35, 38, 43 (doublets), 50, 58 (doublets), 63, and 73 kD. The bands outside the range between 25 and 80 kD were not analyzed. The ocular fluid samples of 10 of the 13 patients stained a band of approximately 28 kD more intense
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FIGURE 1. Immunoblot analysis of a T. gondii crude extract recognized by IgG from serum samples (S) and ocular fluid samples (OF) of 13 patients with ocular toxoplasmosis (the lane numbers correlate with patient numbers given in the Table). Arrows on the right point to the 28-, 30-, and 43-kD bands. Numbers on the left represent molecular weight markers in thousands.
the 28-kD band of the 13 patients was 3.6 (SD 6 3.9). This indicates an overall stronger anti-28-kD response in the eyes of patients with ocular toxoplasmosis. The mean ocular fluid to serum ratio of the 30-kD band was 0.85 (SD 6 0.45), indicating a slightly weaker intraocular anti-30-kD response compared to the response in the serum. The difference in response between the 30- and the 28-kD
than the paired serum samples did. Figure 2 shows a plot of densitometer scans of the lanes of Patient 10 on the immunoblot (within the range of 25 and 80 kD). Staining of the 28-kD band (relative front 5 0.87) and the 30-kD band (relative front 5 0.8) was quantitated as a percentage of total staining and used to calculate an ocular fluid to serum ratio (Figure 3). The mean ocular fluid to serum ratio of 702
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FIGURE 2. Densitometer scans of the lanes of Patient 10 on the immunoblot shown in Figure 1 within the range of 25 to 80 kD. The solid line represents serum; the dotted line represents ocular fluid. The plots of each lane are determined in the x-axis by relative front values (relative front 5 distance of protein migration per length of the lane) and in the y-axis by optical density values. The 30-kD band corresponds to the peaks at relative front 5 0.80 and the 28-kD band corresponds to the peaks at relative front 5 0.87.
bands was found to be statistically significant (P 5 .0175) by using the Wilcoxon signed rank test. A band of 38 kD was stained more intensely by eight of 13 sera compared with staining by paired ocular fluid samples. Bands with approximate molecular weights of 58 and 43 kD were more intensely stained by ocular fluid samples in nine of 13 sample pairs. The samples of the two IgM positive patients did not disclose differences in staining of the above mentioned bands. The control samples from four patients with uveitis from causes other than T. gondii without intraocular anti–T. gondii antibody production but with a sufficiently high titer in ocular fluids all stained the 30-kD band. The samples of Patient 1 also stained a band of apparent molecular weight of 38 kD. No differences in staining patterns between the ocular fluid and the paired serum samples were observed (Figure 4). The ocular fluid to serum ratio of the 30-kD band was 0.90 (SD 6 0.67), and the VOL. 126, NO. 5
ratio of the 28-kD band was 0.94 (SD 6 0.57). No bands were disclosed after immunoblotting with serum from patients without an anti–T. gondii antibody titer or the conjugated antibody alone (data not shown). To investigate whether the reactivity of IgG from ocular fluids against the 28-kD antigen in the crude T. gondii extract was directed to the so-called GRA-2 (granule antigen 2),18 specific IgG from ocular fluid and serum of Patient 10 was absorbed with solid phase bound recombinant GRA-2. After washing and elution (see Methods), eluted IgG was tested with immunoblotting using the T. gondii extract. The eluted serum-IgG only weakly stained at the 28-kD position and had some background staining of the 30-kD band (Figure 5, lane 2), whereas the eluate from the ocular fluid showed evident staining of the 28-kD band (Figure 5, lane 4). The presence of GRA-2 in our T. gondii antigen extract was verified using a monoclonal anti-GRA-2
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FIGURE 3. Graph of relative intensities of the 30- and 28-kD bands of the T. gondii immunoblots as seen in Figure 1. Ratios were calculated of relative intensities of bands per lane of ocular fluid and serum. Ratios higher than 1 indicate a stronger band stained by ocular fluid compared with bands stained by serum.
antibody, which clearly disclosed a strong band at the 28-kD position (Figure 5, lane 5). Solid phase bound glutathione S-transferase, the fusion part of the recombinant GRA-2, was used as a control. After incubation with serum (ocular fluid was no longer available), the eluate did not stain any bands on immunoblot (data not shown). These findings make it very likely that the 28-kD band is identical to GRA-2 and furthermore confirm the observation that ocular fluid of this patient contains relatively more anti-GRA-2 antibodies than the serum does. The same experiment was performed with samples from Patient 13 showing identical results (data not shown).
FIGURE 4. Patients with posterior uveitis caused by an infectious agent other than T. gondii but with anti–T. gondii antibody titers both in the serum and ocular fluid. The infective agents of the inflammation of the eyes are represented at the bottom as follows: 1 5 herpes simplex virus; 2 5 Candida albicans; 3 5 varicella-zoster virus; 4 5 presumed autoimmune uveitis. Numbers on the left represent molecular weight markers in thousands.
patients with ocular toxoplasmosis in this study leads to the supposition that some T. gondii antigens may be more abundant within the eyes of these patients. The paired samples from the two IgM positive patients (with presumed acutely acquired infection) do not disclose these differences in recognition of antigens. These observations suggest a relation between the intraocular antibody response against the 28-, 43-, and 58-kD antigens and a recurrent, chronic infection with T. gondii. Recurrent ocular toxoplasmosis is thought to be due to reactivating cysts within the retina, and it is conceivable that the intraocular immunoresponse is partly directed against antigens from these cysts. Darcy and associates11 showed that a 28.5-kD antigen from a tachyzoite extract was recognized by mouse sera obtained from animals immunized with a
DISCUSSION THE FACT THAT OCULAR TOXOPLASMOSIS IS ASSOCI-
ated with a strong intraocular antibody response has been known for several decades19 and is routinely used for diagnostic purposes. Intraocular toxoplasma antibody testing involves an immunofluorescence technique using intact parasites and thus provides information about the quantity but not the specificity of the parasite antibody response in the eye. The more pronounced recognition of a 28-kD antigen (and to a lesser extent a 43-kD and a 58-kD antigen) by anti–T. gondii IgG from the eyes of most 704
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FIGURE 5. Results of elution experiment with serum and ocular fluid of Patient 10 are shown. After incubation of serum and ocular fluid with solid phase bound rGRA-2 and extensive washing, bound antibodies were eluted in an acid buffer, neutralized, and used for immunoblot analysis of a T. gondii crude extract. Elution samples are 50 times more concentrated. On the bottom, the number 1 represents serum and 3 represents ocular fluid; for eluates of solid phase bound rGRA-2, 2 represents serum and 4 represents ocular fluid; 5 represents monoclonal anti–GRA-2 antibody. Numbers on the left represent molecular weight markers in thousands.
bradyzoite extract. This antigen, later identified as GRA-2,20 is identical to the antigen to which a majority of the ocular toxoplasmosis patients in this study shows a relatively strong intraocular antibody response. This statement is based on absorption and elution experiments with recombinant GRA-2 and the demonstration of the presence of GRA-2 in our crude T. gondii extract using a monoclonal antiGRA-2 antibody. Darcy and associates11 also identified a 43-kD bradyzoite antigen that was recognized by chronic VOL. 126, NO. 5
phase human sera. Whether this antigen, later designated as SAG-3, is similar to the 43-kD band reactivity observed in the study presented here has yet to be established. Of interest is that both the GRA-2 and SAG-3 antigens are expressed by both the tachyzoite as well as the bradyzoite stages of the parasite. This may support the hypothesis that the immunoresponse of patients with chronic recurring ocular toxoplasmosis is driven by bradyzoite antigens leaking from the cysts. Assuming that the initial systemic infection leads to the priming of the immunoresponse against the tachyzoite stage of the parasite, one would assume that a subsequent intraocular immunoresponse driven by antigens leaking from the cysts is possibly directed against antigens shared between bradyzoites and tachyzoites. In the acquired form of the disease, systemic humoral immunity is mainly directed to tachyzoite antigens whereby selection of the intraocular response, when compared with the systemic response, is not yet likely since cyst formation will probably occur later in the disease. Our data showing a similar specificity of the ocular vs the systemic response in two patients with presumed acquired ocular toxoplasmosis would favor this speculative hypothesis. Definitive proof of this hypothesis would involve a comparison of the intraocular immunoresponse with a variety of bradyzoite and tachyzoite antigens. The major surface antigen, SAG-1, with a molecular weight of 30 kD, is considered to be the most abundant surface protein of T. gondii.21 Almost all individuals develop a high serum antibody titer against SAG-1 after exposure to T. gondii; this is one of the earliest, most consistent signs of toxoplasma infection.18,22–24 SAG-1 is the principle antigen recognized by immune antisera from both chronic and acutely-infected individuals.25 The consistent staining of a 30-kD band in our experiments most likely reflects reactivity against SAG-1. Titers obtained with the indirect immunofluorescence assay are probably to a large extent based on the recognition of this surface antigen. Due to the fact that we normalized serum and ocular fluid samples with respect to their immunofluorescent assay anti–T. gondii titers, the 30-kD band on the immunoblots of most paired samples stained with similar intensities.
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10. Kijlstra A, Rothova A, Baarsma GS, et al. Computer registration of uveitis patients. Doc Ophthalmol 1987; 67:139 –143. 11. Darcy F, Charif H, Caron H, et al. Identification and biochemical characterization of antigens of tachyzoites and bradyzoites of Toxoplasma gondii with cross-reactive epitopes. Parasitol Res 1990;76:473– 478. 12. Van der Lelij A, Rothova A. Diagnostic anterior chamber paracentesis in uveitis: a safe procedure? Br J Ophthalmol 1997;81:976 –979. 13. Mancini G, Carbonara AO, Heremans JF. Immunochemical quantitation of antisera by single radial immunodiffusion. Immunohistochemistry 1965;2:235–256. 14. Goldmann H, Wittmer R. Antikorper im kammerwasser. Ophthalmologica 1954;127:323–230. 15. Luyendijk L, de Boer JH, Rothova A, et al. Sensitivity and specificity of anti-viral antibody determination in the aqueous or vitreous of uveitis patients. In: Dernouchamps JP, Verougstreate I, Caspers-Velu I, Tassignon MJ, editors. Recent advances in uveitis. Amsterdam: Kugler, 1993:265– 267. 16. Bessieres MH, Le Breton S, Seguela JP. Analysis by immunoblotting of Toxoplasma gondii exo-antigens and comparison with somatic antigens. Parasitol Res 1992;78: 222–228. 17. Parmley SF, Sgarlato GD, Mark J, Prince JB, Remington JS. Expression, characterization, and serologic reactivity of recombinant surface antigen P22 of Toxoplasma gondii. J Clin Microbiol 1992;30:1127–1133. 18. Potasman I, Araujo FG, Desmonts G, Remington JS. Analysis of Toxoplasma gondii antigens recognized by human sera obtained before and after acute infection. J Infect Dis 1986;154:650 – 657. 19. Desmonts G. Definitive serological diagnosis of ocular toxoplasmosis. Arch Ophthalmol 1966;76:839 – 851. 20. Parmley SF, Sgarlato GD, Remington JS. Genomic and corrected cDNA sequence of the P28 gene from Toxoplasma gondii. Mol Biochem Parasitol 1993;57:161–165. 21. Kasper LH, Crabb JH, Pfefferkorn ER. Purification of a major membrane protein of Toxoplasma gondii by immunoabsorption with a monoclonal antibody. J Immunol 1983; 130:2407–2412. 22. Remington JS, Araujo FG, Desmonts G. Recognition of different Toxoplasma antigens by IgM and IgG antibodies in mothers and their congenitally infected newborns. J Infect Dis 1985;152:1020 –1024. 23. Santoro F, Afchain D, Pierce R, Cesbron JY, Ovlaque G, Capron A. Serodiagnosis of toxoplasma infection using a purified parasite protein (P30). Clin Exp Immunol 1985; 62:262–269. 24. Sharma SD, Mullenax J, Araujo FG, Erlich HA, Remington JS. Western Blot analysis of the antigens of Toxoplasma gondii recognized by human IgM and IgG antibodies. J Immunol 1983;131:977–983. 25. Decoster A, Darcy F, Caron A, Capron A. IgA antibodies against P30 as markers of congenital and acute toxoplasmosis. Lancet 1988;2:1104 –1107.
Since all patients in our study were reported as a diagnostic dilemma and the number of patients was small, our results may only be reflective of this selected group. Future studies are required to draw more general conclusions about the intraocular antibody response and may improve our knowledge concerning the pathogenesis of ocular toxoplasmosis, which can lead to further refinement of the laboratory diagnosis of this disease. ACKNOWLEDGMENT
We wish to thank members of the Dutch Uveitis Study Group for their help concerning patient samples and clinical information. We thank Dr T. van Gool from the Department of Microbiology, University of Amsterdam, for providing us with the T. gondii tachyzoites.
REFERENCES 1. Nussenblatt RB, Palestine AG. Ocular toxoplasmosis. In: Marshall DK, editor. Uveitis: fundamentals and clinical practice. Chicago: Yearbook Medical Publishers, 1989: 336 –354. 2. Loewer-Sieger DH, Rothova A, Koppe JG, et al. Congenital toxoplasmosis: a prospective study based on 1821 pregnant women. In: Saari KM, editor. Uveitis update. Amsterdam: Elsevier, 1984:203–207. 3. Frenkel JK. Pathophysiology of toxoplasmosis. Parasitol Today 1988;4:273–278. 4. Glasner PD, Silveira C, Kruszon-Moran D, et al. An unusually high prevalence of ocular toxoplasmosis in southern Brazil. Am J Ophthalmol 1992;114:136 –144. 5. Ronday MJ, Luyendijk L, Baarsma GS, Bollemeijer JG, Van der Lelij A, Rothova A. Presumed acquired ocular toxoplasmosis. Arch Ophthalmol 1995;113:1524 –1529. 6. Ambroise-Thomas P, Pelloux H. Toxoplasmosis— congenital and in immunocompromised patients: a parallel. Parasitol Today 1993;9:61– 63. 7. Frenkel JK. Host, strain and treatment variation as factors in the pathogenesis of toxoplasmosis. Am J Trop Med Hyg 1953;2:390 – 415. 8. Gazzinelli RT, Denkers EY, Sher A. Host resistance to Toxoplasma gondii: model for studying the selective induction of cell-mediated immunity by intracellular parasites. Infect Agents Dis 1993;2:139 –149. 9. Kijlstra A, Luyendijk L, Baarsma GS, et al. Aqueous humor analysis as a diagnostic tool in toxoplasma uveitis. Int Ophthalmol 1989;13:383–386.
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