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A Lytic Monoclonal Antibody to Trypanosoma cruzi Bloodstream Trypomastigotes Which Recognizes an Epitope Expressed in Tissues Affected in Chagas' Disease NORBERTO W. ZWIRNER, EMILIO L. MALCHIODI,* MONICA G. CHIARAMONTE, AND CARLOS A. FOSSATI
Instituto de Estudios de la Inmunidad Humoral, Cdtedra de Inmunologia, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Buenos Aires, Argentina Received 13 September 1993/Returned for modification 2 November 1993/Accepted 4 March 1994
It has been suggested that molecular mimicry between the antigens of Trypanosoma cruzi and the host could have a role in the onset of the chronic stage of Chagas' disease. In this article, we report on a monoclonal antibody (MAb), CAK20.12 (immunoglobulin G2b), which reacts with a polypeptidic epitope of a 150-kDa antigen expressed on the surface of several strains of T. cruzi. This MAb also causes lysis of bloodstream trypomastigotes. Serum samples from 30 of 30 patients with chronic and 11 of 13 patients with acute Chagas' disease present specific antibodies to this antigen. MAb CAK20.12 reacts, by indirect immunofluorescence, with human and syngeneic murine striated muscle tissue, with the smooth muscle layer of cardiac arteries, with the lamina muscularis mucosae and the external striated muscle layer of the esophagus, and with the smooth muscle cells of the colon from normal syngeneic mice. Reactivity with the small intestine was very weak, and no reactivity with ventricle or atrium tissue was detected. Adsorption with an antigenic fraction from normal murine striated muscle or from T. cruzi epimastigotes confirmed that MAb CAK20.12 recognizes a common epitope present in parasites and host tissues. MAb CAK20.12, lytic for the infective form of T. cruzi, recognizes an epitope expressed in striated and smooth muscle cells of the host tissues affected in the chronic stage of Chagas' disease. American trypanosomiasis, or Chagas' disease, is caused by Trypanosoma cruzi. The disease has two clinically distinct stages: the acute stage is characterized by the presence of trypomastigotes in blood, and in the chronic stage, the parasitemia is very low, detection being possible in only about 50% of patients by xenodiagnosis or hemoculture. Different types of cardiopathy may appear in this stage, such as mild arrhythmia, right or left branch block, and/or severe myocardiopathies which can cause death. Disorders of the esophagus and/or colon (megaviscera) may also be present in other patients. The humoral immune response to T. cruzi infection is complex. Some of the antibodies (Abs) which can be detected in the sera of naturally infected human beings or experimentally infected laboratory animals are lytic for the parasite. This type of Ab has not been found in the sera of animals immunized with different antigens (Ags) of the parasite (22). Lytic Abs have been thought to have a protective role against the disease in murine experimental models (18, 21, 22, 35), although it was not pointed out whether the immunoprotective
the immune system and giving rise to an autoimmune response (17). Other authors suggest that T. cruzi shares Ags with the host and that the effector elements produced as a response to infection recognize their own Ags (molecular mimicry) (5, 14, 30). Some homologous Ags between T. cruzi and mammals have been described, such as some ribosomal proteins (23, 32), laminin (34), tubulin (1, 26), a muscle sarcoplasmic Ag (28), and some Ags of the nervous system (33, 39, 43). Antibodies against these Ags have been found in the serum of individuals with Chagas' disease. Many monoclonal antibodies (MAbs) have been obtained, making possible the study of the Ags of T. cruzi which may be important for diagnosis (6, 15, 45) and the Ags which participate in different processes of the host-parasite relationship (27, 29, 31, 37, 44). The production of an MAb which recognizes a 150-kDa Ag in T. cruzi and provokes lysis of trypomastigotes is described in this article. The MAb also recognizes an Ag present in the soluble sarcoplasmic fraction of normal murine striated muscle and in the smooth muscle of the cardiac arteries, esophagus, and colon of the host.
effects were able to avoid or attenuate the chronic stage of the disease. The origin of the tissue injuries characteristic of the chronic stage has been a controversial issue. Different mechanisms have been proposed to explain the immunopathology of the chronic disease; e.g., that the Ags released by the parasite could be adsorbed on normal cells of the host, making them a target for the antiparasite response (42). Lysis of the host cell would release the cell's own Ags, leading to their contact with
MATERIALS AND METHODS Parasites. (i) Epimastigotes. T. cruzi epimastigotes from strains (Tulahuen, RA, CAI, AF, AWP, LP, and Mesa) and the k98 clone of the CA1 strain were cultured in a biphasic medium. The parasites were collected and processed to obtain live or Formalin-fixed epimastigotes as described previously (45). (ii) Trypomastigotes. Bloodstream trypomastigotes (RA strain) were isolated from the blood of acutely infected mice. To this end, Rockland mice, 23 ± 1 days of age, intraperitoneally infected 1 week earlier with 5 x 105 blood forms were seven
* Corresponding author. Mailing address: Instituto de Estudios de la Inmunidad Humoral (IDEHU-CONICET), Catedra de Inmunologia, FFyB, Junin 956, 4°P, 1113 Buenos Aires, Argentina. Phone: 54-1-9613021. Fax: 54-1-962-5341. Electronic mail address:
[email protected]
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bled, and the parasites were isolated by differential centrifugation. Blood was centrifuged for 1 min at 500 x g, and the tube was incubated at 37°C for 1 h. The supernatant containing the parasites was separated, and the trypomastigotes were washed with a solution containing 137 mM NaCl, 5 mM KCl, 12 mM glucose, and 15 mM phosphate, pH 7.4 (GKN). Trypomastigotes were used in a complement-mediated lytic assay and in indirect immunofluorescence. For the isolation of trypomastigotes of the CA, strain of T. cruzi, the same protocol was used but the infected mice were bled on day 25 postinfection (time of parasitemia peak). Antigenic fractions of epimastigotes. One gram (wet weight) of washed epimastigotes was frozen and thawed three times and then suspended in 2.65 ml of 0.25 M sucrose-5 mM KCl with protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 5 ,uM leupeptin, S FM E-64, and 5 ,uM pepstatin [Sigma]). After centrifugation for 10 min at 6,000 x g at 40C, the supernatant (Si) was kept on ice. The pellet was suspended in the same solution and centrifuged for 10 min at 17,000 x g at 4°C. This supernatant (S2) was pooled with SI and centrifuged for 30 min at 45,000 x g at 4°C. The resulting supernatant was called F45. Protein content was determined by the method of Bradford (4). Infection of BALB/c mice. BALB/c mice were intraperitoneally infected with 105 bloodstream trypomastigotes of the k98 clone of the CA, strain of T. cruzi. Seventy-five days later, the spleen was removed and used for the production of hybridomas. MAbs to T. cruzi. MAb CAK20.12 (immunoglobulin G2b [IgG2b]) was derived by somatic cell hybridization as described by Kohler and Milstein (20), with polyethylene glycol as the fusogenic agent. NSO myeloma cells were fused with spleen cells from infected mice, and the resulting hybridomas were cloned by limiting dilution. The screening assay was performed by an indirect immunoenzymatic method (enzyme-linked immunosorbent assay [ELISA]) with F45 as the antigen. The isotype of the MAb was determined by double diffusion of culture supernatants with commercial anti-isotypic sera. The Ab concentration of ascitic fluid was determined by densitometric scanning of an electrophoretic proteinogram. Purification of MAb CAK20.12. MAb CAK20.12 was purified from ascitic fluid by anion-exchange fast protein liquid chromatography (FPLC) with an XK16/40 column packed with S-Sepharose (LKB-Pharmacia). A discontinuous gradient over 30 min of 50 mM MES (morpholineethanesulfonic acid)-20 mM NaCl, pH 5.5 (buffer A) and 50 mM MES-1 M NaCl, pH 5.5 (buffer B) at 10 ml/min was used. The collected fractions were lyophilized. Purity was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Ab activity was determined by an indirect ELISA with F45 adsorbed onto microtiter plates. Amplification ELISA. The experimental design of Voller et al. (41) was used for the amplification ELISA. Ten micrograms of F45 per milliliter was adsorbed onto Immulon 2 (Dynatech) microtiter plates. After being blocked with BS solution (phosphate-buffered saline [PBS] containing 3% bovine serum albumin [BSA; Sigma] and 0.1% gelatin [Merck]), the plates were washed with PBS containing 0.05% Tween 20 (PBST). Hybridoma supernatants were assayed undiluted. After being washed, rabbit immunoglobulins to mouse immunoglobulins, coupled to horseradish peroxidase (DAKO), diluted 1:1,000 in 0.1% gelatin in PBST, were used as the second antibody. The first and second antibodies were incubated for 1 h at 37°C. After the wells were washed, the content of each well was developed with a solution containing o-phenylenediamine (1 mg/ml; Merck) and 30% H202 (1 ,ul/ml) in 0.1 M citrate-phosphate
buffer, pH 5. The reaction was stopped with 2 M H2SO4 (30 ,I per well). The resulting color was read at 490 nm in an ELISA reader (Metertech). Capture ELISA. For the capture ELISA, FPLC-purified MAb CAK20.12 (2 jig per well) was adsorbed onto Immulon-2 plates (Dynatech). After being blocked with BS solution, the plates were incubated with F45 (10 jig per well) in PBS containing 1% BSA, 0.1% gelatin, and 0.05% Tween 20 for 1 h at 37°C. After being washed, the human sera under study, diluted 1:400 in the same solution, were incubated for 30 min at room temperature. The plates were washed, and each well was incubated with 50 jl of anti-human IgG MAb-horseradish peroxidase conjugate diluted 1:8,000 for 45 min at room temperature. The content of each well was developed as described above. Control experiments for nonspecific adsorption of Ag with a control capture MAb (BI24 [10]) and without addition of the capture MAb were performed throughout. Each serum was also tested in the absence of Ag to detect anti-murine IgG activity. The cutoff value was calculated as the mean optical density plus 3 standard deviations of the sera from the healthy controls. IIF with parasites. Formalin-fixed trypomastigotes (RA strain) and epimastigotes of several strains were used for indirect immunofluorescence (IIF) as described by Alvarez et al. (2). Ascitic fluid of MAb CAK20.12 diluted 1:30 was assayed. As the second antibody, rabbit anti-mouse immunoglobulins coupled to fluorescein isothiocyanate was used. The slides were observed in a Zeiss standard 14 IFD epifluorescence microscope. For IIF tests with live epimastigotes and trypomastigotes, 106 parasites were incubated for 30 min at 4°C with 0.1 ml of a 1:30 dilution of the ascitic fluid of MAb CAK20.12 in the presence of 0.2% sodium azide. After being washed, the epimastigotes were air dried onto glass slides and fixed with cold acetone for 2 min. Incubation with the second antibody and the rest of the assay were performed as described above. An isotype-matched anti-Brucella abortus MAb (BI24 [10]) was used as a negative control. IIF with mammalian tissues. Normal BALB/c mice (60 days old, pathogen-free) were killed by cervical dislocation, and the thighs, heart, esophagus, small intestine, and colon were excised. The organs and tissues were mounted in 10.24% (wt/wt) polyvinyl alcohol, 4.26% Carbowax, and 85.5% (wt/wt) nonreactive material (OCT embedding compound; Histological Equipment Ltd.) and frozen in liquid nitrogen. Sections 4-jim thick were cut in a cryostat (Minotome), mounted onto glass slides, air dried, and fixed with cold acetone for 4 min. The slides were kept at -70°C until use. IIF tests were performed as described above, but the first and second antibodies were diluted in the presence of 3% heat-aggregated normal rabbit immunoglobulins to minimize background staining.
IIF tests of human striated muscle tissue were also performed on sections of a piece of the thigh of an amputated leg from a healthy human who suffered an automobile accident. The assay was carried out as described above except that the first and second antibodies were diluted in the presence of 10% normal human serum instead of 3% heat-aggregated normal rabbit immunoglobulins. Adsorption of MAb CAK20.12 with antigenic fractions of T. cruzi epimastigotes and striated muscle tissue. The F45 fraction of epimastigotes and an extract of striated muscle obtained by homogenization of thigh tissue with distilled water were lyophilized. Both fractions were used for the adsorption of purified MAb CAK20.12 for 1 h at room temperature. The supernatants were used in the IIF tests as described above.
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Complement-mediated lytic assay of trypomastigotes. A suspension of 2 x 107 to 4 x 107 bloodstream trypomastigotes per ml, obtained from infected mice, was incubated with ascitic fluid or FPLC-purified MAbs for 30 min at 4°C. One volume of fresh normal guinea pig serum was added, and the tubes were incubated for 1 h at 37°C. Motile live parasites were counted in a Neubauer chamber, and the percent lysis was calculated with reference to the tube with no antibody (0% lysis). Intact but nonmotile trypomastigotes were not observed. All experiments were performed in duplicate. SDS-PAGE. SDS-PAGE was performed by the method of Hames (13) with 7.5% acrylamide gels. In some cases, the samples were heated for 3 min at 100°C. Some gels were stained with Coomassie blue R-250. Immunoblotting. Immunoblotting analysis was performed as described by Tsang et al. (38). Nitrocellulose sheets were blocked with 0.5% skim milk, and FPLC-purified MAbs were assayed at 50 pLg/ml. As the secondary antibody, rabbit antimouse immunoglobulins coupled to horseradish peroxidase were used. The reaction was developed with 4-chloro-1-naphthol (0.37 mg/ml) and 30% H202 (0.5 ,ul/ml). Fractions of striated muscle tissue. Soluble sarcoplasmic proteins and myofibrils were prepared as described by Goll and Robson (11). For this purpose, the thighs of normal BALB/c mice were homogenized in an Ultra Turrax tissue homogenizer with 0.25 M sucrose (Mallinckrodt)-1 mM EDTA (Mallinckrodt)-0.05 M Tris-HCl (BDH), pH 7.6. The extract was stirred for 30 min, and the resulting suspension was centrifuged for 10 min at 2,500 x g. The pellet was suspended in the above solution, stirred for 15 min, and centrifuged for 10 min at 2,500 x g. Both supernatants were pooled and frozen. This fraction contains all the soluble sarcoplasmic proteins. The pellet was suspended in 1 mM EDTA-0.05 M Tris-HCl, pH 7.6. The connective tissue was removed, and the myofibrils were purified by successive cycles of suspension-centrifugation in (i) 0.15 M KCl-0.03 M Tris-HCl (pH 7.6), (ii) 1 mM EDTA (pH 7.6), (iii) distilled water, (iv) 0.15 M KCl-0.03 M Tris-HCl (pH 7.6), and (v) 0.15 M KCI-0.03 M Tris-HCl (pH 7.6). Purified myofibrils were partially solubilized in 0.6 M KCl-0.03 M Tris-HCl, pH 7.6. Protein content was determined by the method of Bradford (4). Periodate oxidation of antigenic fractions. The F45 fraction of epimastigotes was oxidized with sodium periodate as described by Vennegoor et al. (40). Briefly, 1 ml of the antigenic fraction, dialyzed against 0.1 M acetic acid-sodium acetate (pH 4.4), was incubated with 35 [l of a 2 M solution of sodium m-periodate (Mallinckrodt) for 18 h at 4°C. Thirty-five microliters of a 1:5 solution of glycerin was added, and the solution was dialyzed against PBS and frozen until use. Digestion of antigenic fractions with proteinase K. The F45 fraction of epimastigotes was incubated overnight at 37°C with proteinase K (Sigma) and 0.2% sodium azide, at a ratio of protein to enzyme of 100:1. The digested fraction was frozen until use. The effect of the sodium azide on the proteolytic activity of the proteinase K had been previously assayed. Dot-blot. The F45 fraction of epimastigotes, the sarcoplasmic fraction, and the myofibrils of striated muscle (whole fractions, periodate degraded or proteinase K digested) were adsorbed onto nitrocellulose papers (20,ug per spot) with a dot-blot apparatus (Bio-Rad). The blocking step, washing procedure, and incubation with antibodies were performed as described for immunoblotting. Human sera. Serum samples from 30 patients with chronic Chagas' disease were studied. The cases were classified as chronic by the absence of parasitemia and positive conventional serological tests (direct agglutination of Formalin-fixed
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FIG. 1. Complement-mediated lysis of bloodstream trypomastigotes of T. cruzi (RA strain). FPLC-purified MAb CAK20.12 (solid bars) and MAb B124 (anti-B. aborttis negative control; hatched bars) and their respective ascitic fluids (open bars, MAb CAK20.12; stippled bars, MAb B124) were assayed. Purified MAbs were used at 2.5 mg/ml (bars 1), 1.25 mg/ml (bars 2), and 0.625 mg/ml (bars 3). Ascitic fluids (Ab concentration, 5 mg/ml) were used undiluted (bars 4) or diluted 1:2 (bars 5) or 1:4 (bars 6). Error bars show the standard deviation.
epimastigotes with titers greater than 1:32, indirect hemagglutination with titers greater than 1:64, and/or IIF with Formalinfixed epimastigotes with titers greater than 1:30). Some of the patients had chronic chagasic pathologies, such as electrocardiographic abnormalities, heart arrhythmia, severe myocardiopathies, or megaviscera. Moreover, 13 serum samples from acute chagasic patients were studied. These patients had parasitemia as detected by xenodiagnosis, hemoculture, or microhematocrit. Some of them also had positive conventional serological tests. Serum samples from healthy volunteers negative for T. cruzi Ags by serological tests were used as negative controls.
RESULTS
Reactivity of MAb CAK20.12 with T. cruzi strains. MAb CAK20.12 (IgG2b) was derived from the spleen of a BALB/c mouse chronically infected with the k98 clone of the CA, strain
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FIG. 2. Reactivity of human sera in a capture ELISA with MAb CAK20. 12. Each point represents the optical density (OD) resulting from subtracting the absorbance of the well without Ag from that of the well with Ag for sera from healthy volunteers (NHS) and patients with acute chagasic (AChS) and chronic chagasic (CChS) disease. The cutoff value was calculated as the mean absorbance of the negative-control sera plus 3 standard deviations and is indicated by a horizontal dotted line.
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FIG. 3. IIF on sections of normal BALB/c mouse tissues. (A) Longitudinal section of striated muscle tissue stained with MAb CAK20.12; three cells showing a pattern of cytoplasmic fluorescence distributed longitudinally, paralleling the myofibril array and the sarcoplasmic reticulum in the muscle cell, can be observed. Fluorescence of the cell membrane was also detected. Magnification, X400. (B) Section of heart tissue stained with MAb CAK20.12, showing fluorescence associated with the smooth muscle layer of the cardiac artheries. v, ventricle; a, atrium. Magnification, x250. (C) Section of esophagus stained with MAb CAK20.12; fluorescence is located in the smooth muscle layer of the mucous layer (lamina muscularis mucosae) and in the organ outer layer. m, mucous layer; mm, lamina muscularis mucosae; sm, submucous layer; em, external muscular layer. Magnification, x250. (D) Longitudinal section of striated muscle tissue stained with MAb BI24 (negative control). Magnification, x400. (E) Section of heart tissue stained with MAb B124. v, ventricle; a, atrium. Magnification, x250. (F) Section of esophagus stained with MAb BI24. m, mucous layer; mm, lamina muscularis mucosae; sm, submucous layer; em, external muscular layer. Magnification, X250.
of T. cruzi. This MAb reacted by IIF with RA trypomastigotes, the fluorescence being detected mainly in the parasite membrane. Fluorescence was found to be uniformly distributed throughout the parasite in epimastigotes of different T. cruzi strains (Tulahuen, RA, AWP, LP, Mesa, AF, CA,, and its k98 clone). With live epimastigotes and trypomastigotes, fluorescence was seen only on the cell membrane (results not shown). Lytic activity on blood trypomastigotes. Since CAK20.12 recognized a surface Ag of the parasite, its ability to lyse blood forms in the presence of guinea pig complement was analyzed. The results obtained showed that MAb CAK20.12 lysed 90.2% ± 5.4% and 88.5% ± 8% of trypomastigotes of the CA, and RA strains, respectively (Fig. 1). This effect depended on the Ab dose and could only be detected when purified MAb was used. No significant lysis was observed when ascitic fluid was used in spite of equivalent Ab concentrations. No lysis was ever found in the negative controls. Reactivity of human sera with the Ag recognized by MAb
CAK20.12. The reactivity of sera from patients infected with T. cruzi was studied with purified MAb CAK20.12 by means of a capture ELISA of the F45 fraction. It was shown that 100% (30 of 30) of the samples from patients with chronic Chagas' disease and 85% (11 of 13) of those from patients with acute Chagas' disease exhibited a reactivity higher than the mean plus 3 standard deviations for sera from healthy controls (Fig.
2). IIF of mouse tissue sections. MAb CAK20.12 reacted with normal syngeneic striated muscle tissue. Figure 3A shows a pattern of cytoplasmic fluorescence distributed longitudinally, paralleling the myofibril array and the sarcoplasmic reticulum in the muscle cell. Fluorescence of the cell membrane was also detected. It was observed that CAK20.12 reacts with the smooth muscle layer of the cardiac arteries when sections of heart tissue of normal mice were used (Fig. 3B). Figure 3C shows the results obtained with esophagus sections from a normal syngeneic mouse. Fluorescence is located in the
MAb TO T. CRUZI TRYPOMASTIGOTES AND HOST TISSUES
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FIG. 4. IIF of trypomastigotes of T. cruzi with MAb CAK20.12. (A) IIF of parasites stained with FPLC-purified MAb, showing a fluorescence pattern along the parasite membrane. Magnification, X400. (B) IIF of trypomastigotes stained with FPLC-purified MAb preadsorbed with an extract of normal mouse striated muscle tissue. Fluorescence was abolished by adsorption of the MAb with an antigenic fraction of striated muscle from a normal mouse. Magnification, x400.
B
smooth muscle layer of the mucous layer (lamina muscularis mucosae) and in the organ outer layer. The same studies performed on normal mouse colon (not shown) exhibited fluorescence associated with the outer smooth muscle layer. On the other hand, very weak reactivity was detected in small intestine sections. Sections of syngeneic ventricle or atrium were negative (not shown). Significant fluorescence was never detected with the control MAb. Similar results were consistently obtained with tissues from three mice. MAb CAK20.12 also reacted with normal human striated muscle tissue; a cytoplasmic fluorescence similar to that shown in Fig. 3A for murine striated muscle was observed (not shown). Adsorption of MAb CAK20.12 with an antigenic fraction of striated muscle from a normal mouse inhibited fluorescence on either epimastigotes (not shown) or trypomastigotes of T. cruzi (Fig. 4). Moreover, adsorption of the MAb with the F45 fraction of epimastigotes inhibited the fluorescence on muscle tissue (not shown). Reactivity of MAb by dot-blot and Western blot. Figure SA shows that MAb CAK20.12 reacts by dot-blot with the F45 fraction of the parasite. The same holds true for the soluble sarcoplasmic fraction of normal murine striated muscle, whereas no reaction was observed with purified myofibrils. No reactivity with any of the Ags studied was detected with the control MAb. Degradation with proteinase K of the F45 antigenic fractions of T. cruzi epimastigotes caused loss of reactivity of MAb CAK20.12 by dot-blot. Conversely, chemical oxidation of the carbohydrates of the same antigenic fraction caused no change in the recognition of the Ag by CAK20.12 (Fig. 5B). By Western blot (immunoblot) of the F45 fraction of T. cruzi epimastigotes, MAb CAK20.12 recognized a 150-kDa component. Heating of the antigenic fraction at 100°C for 3 min in SDS-PAGE sample buffer abolished the reactivity of the MAb
C kDa
(Fig. SC). DISCUSSION
MAb CAK20.12 (IgG2b) was derived from the spleen of a mouse chronically infected with the k98 clone of the CA, strain of T. cruzi. This MAb recognizes an Ag that is widely distributed among strains of T. cruzi, and from the IIF results with both Formalin-treated trypomastigotes and live epimastigotes and trypomastigotes, it is located on the surface of T. cruzi. It has been demonstrated by Western blotting of the epimasti-
B124 CAK20.12 1 2 3 4 5
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FIG. 5. Reactivity of MAb CAK20.12 by dot-blot and Western blot. (A) F45 fraction of epimastigotes of T. cruzi (lanes 1 and 2), the sarcoplasmic fraction of normal mouse striated muscle cells (lanes 3 and 4), and myofibrils (lanes 5 and 6) were assayed with MAb CAK20.12 (lanes 1, 3, and 5) and MAb B124 (negative control; lanes 2, 4, and 6). (B) The untreated F45 fraction (lane 1), F45 digested with proteinase K (lane 3), and F45 oxidized with periodate (lane 5) were assayed with the MAbs; mock-treated F45 fraction was also assayed (proteinase K treatment, lane 2; periodate treatment, lane 4). (C) Western blot of F45 fraction with MAb CAK20.12 (lanes A and B) and MAb B124 (lanes C and D). Nonboiled (lanes B and D) and boiled (lanes A and C) F45 fractions were also assayed.
gote lysate that MAb CAK20.12 recognizes an epitope present in an Ag of 150 kDa (Fig. 5). This epitope is sensitive to the heating undergone by samples to be used in SDS-PAGE, being modified in such a way that it can no longer be recognized by its specific Ab. The epitope recognized might be a polypeptide, since it is damaged by proteinase K treatment but unaffected by chemical oxidation with sodium periodate (Fig. 5). Several authors have reported that either natural or experimental infection with T cruzi triggers the synthesis of lytic antibodies (18, 22). Particularly with clone k8 of the CA, strain, the sera of infected mice have a low lytic activity and a slight opsonization effect on trypomastigotes (7). This strain, as well as its clone k98, is nonlethal for that host (12). By using that strain for infection, we have obtained an MAb with lytic activity not only for the infective form of the homologous strain (CA,) but also for trypomastigotes of the RA strain, which is highly lethal for mice. In spite of the different T. cruzi trypomastigote components described, which would provide them with the ability to avoid its lysis by Ab and complement
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(8, 9, 16, 19, 36), the percentages of lysis are high (about 90% for both strains). Some anti-T. cruzi lytic MAbs have been described (27, 29, 31, 44), but none of them appear to recognize Ags related to the one recognized by MAb CAK20.12. Norris et al. (25) described a 160-kDa glycoprotein expressed in metacyclic trypomastigotes but not detected in epimastigotes. This Ag, when injected into rabbits, induces the synthesis of lytic antibodies. Although this Ag is similar in molecular weight to the one described here, the Ag recognized by MAb CAK20.12 is easily detected in epimastigotes. This leads us to consider that the two Ags are different, but further studies are needed to demonstrate this possibility. CAK20.12 is of an isotype that is able to fix complement, but it was only possible to obtain a significant degree of lysis when the purified MAb was used, since no lysis could be detected when ascitic fluid was used (Fig. 1). This result can be explained by the presence of inhibitors of complement fixation in ascitic fluid, previously described by Appelmelk et al. (3). The Ag recognized by MAb CAK20.12 would be highly immunogenic during natural infection in humans, since specific Abs were detected in all of the sera from patients with chronic Chagas' disease as well as in 85% of sera from patients with acute Chagas' disease (Fig. 2). These results suggest that the Ag recognized by MAb CAK20.12 is an immunogenic target for those Abs which induce complement-mediated lysis of the blood forms of T. cruzi. That Ag could be involved in the mechanism of resistance to live parasites. Because of the myotropic feature of the infective strain used for the production of CAK20.12 (12) and of the present controversial existence of autoimmunity in the chronic stage of Chagas' disease (14, 17, 30), we have analyzed the reactivity of the MAb against normal mouse tissue. It was noticed that CAK20.12 recognizes an epitope present in striated muscle of syngeneic mice (Fig. 3A). Taking into account the fluorescence distribution, it can be assumed that the epitope is present in an Ag that is distributed longitudinally in the cell, parallel to the distribution of myofibrils and the sarcoplasmic reticulum within the muscle cell. Studies performed on heart (Fig. 3B), esophagus (Fig. 3C), and colon led to the conclusion that MAb CAK20.12 also recognizes an epitope present in the smooth muscle tissue of the cardiac arteries and the esophagus and colon wall. At variance with this, the epitope was absent in the small intestine, atrium, and ventricle of syngeneic animals. Thus, the MAb would recognize an epitope present in an Ag expressed by the muscle cells of the tissues usually affected in the chronic stage of the disease. The fact that the reactivity detected could be due to a heterophilic Ab can be ruled out, since only sections of syngeneic tissues have been used. The epitope is also present in human cells, since MAb CAK20.12 reacts with normal striated muscle tissue, exhibiting a fluorescence pattern similar to that seen with murine striated muscle tissue (not shown). The results of cross-adsorption with normal striated muscle (Fig. 4) and with F45 of epimastigotes confirm the finding that MAb CAK20.12 recognizes an epitope present in both T. cruzi and host muscle tissue. The epitope is found in the soluble sarcoplasmic fraction (Fig. 5); it would not be part of a muscle structural protein, such as myosin or actin (24, 26). Some authors have reported the production of MAbs which react both with components of the parasite and with host tissues. An MAb against a subpopulation of rat neurons which reacts with epimastigotes and amastigotes of T. cruzi has been described (43). Other authors have obtained an anti-T. cruzi MAb having the ability to react with central and peripheral
neurons of mice and rats (33). Cultured parasites were used in those studies instead of blood trypomastigotes, which was criticized by Kierszenbaum (17). The fact that MAb CAK20.12 could recognize any of the components of the medium used for epimastigote culture cannot be suspected in our study, since this MAb also reacts with trypomastigotes isolated from the blood of infected mice, which have never been in contact with the components of the culture medium. Moreover, the results obtained in the studies of cross-adsorption with both host tissue and parasites are conclusive. MAb CAK20.12, lytic for the infective form of T. cruzi, recognizes an epitope present in both smooth and striated muscle cells of host tissues involved in chronic chagasic pathology.
ACKNOWLEDGMENTS Our thanks to Stella Maris Gonzalez Cappa for supplying the infected mice, Elvira D. de Isola and Estela Lammel for epimastigote cultures, and Gerardo Mirkin for assistance in making cryostat sections. This work was supported by grants from CONICET and UBA. Norberto W. Zwirner and M6nica Chiaramonte are Fellows of CONICET. Emilio L. Malchiodi and Carlos A. Fossati are members of the Research Career of CONICET. C.A.F. is also a member of the Catedra de Inmunologia, Facultad de Ciencias Exactas, Universidad
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