Immunoperoxidase technique in experimental chronic chagasic myocarditis

Abstracts

Chagas'disease has been described as the commonest form of chronic myocarditis. An immunologic pathogenesis has been discribed for this form of the disease. So far, no immunoperoxidase technique has been used for the detection of immunological deposits in chronic experimental Chagas'myocardiopathy. Forty-one Swiss mice, three months old were inoculated intraperitoneally with doses between 10 and 10(5) Tulahuen trypomastigotes. Mice were reinoculated one month after with doses between 10² and 10(5) and sacrificed at 6 (n=21) and 9 months (n=9) after the first inoculation. ECGs were recorded before sacrifice. Immunoperoxidase technique (peroxidase-antiperoxidase method), immunofluorescence (direct and indirect) as well as histological studies were performed in myocardiums and skeletal muscles of the surviving animals. The most sensitive methods for detecting chronic chagasic infection were the routine histologic studies (73%) and the ECGs 83% and 89% on 6 and 9 mo. post-infected mice, respectively. Myocardial involvement varied from interstitial mild focal lymphocyte infiltrates up to replacement of myocytes by loose connective tissue. Atrial myocardiums (21/23, 91%) were more affected than ventricles (9/23, 39%). Typical chagasic nests were rarely found. Skeletal muscle involvement (11/18 and 7/9) varied from mild to extensive lymphocyte and plasmacell infiltrates, and necrotic fibers. The involved antigen were shown in skeletal muscles by the immunoperoxidase technique as diffusely arranged granular intracytoplasmatic deposit for both IgC and total immunoglobulins. The coincidence between this technique and histologic muscle lesions was 11/18 (61(%) in 6 mo. and 6/8 (75%) at 9 mo. post-infection. In heart, delicate granular deposits of total immunoglobulins were seen diffusely arranged within the ventricular myocytes; coincidence between immunoperoxidase technique anl histologic involvement increased from 36 to 66% in animals sacrifeced 6 and 9 mo. post-infection. This strongly stressed the increase of immunologic phenomena with the chronification of infection. Concerning sensitivity, immunoperoxidase and direct immunofluorescence were highly sensitive in skeletal muscle (100%, p < 0.01). Conversely, direct immunofluorescence technique showed poor results in heart while immunoperoxidase increased its sensitivity from 21.4% (at 6 mo.) to 66.6% (at 9 mo.) post-infection (p < 0.001). Considering the necessity of obtaining an adequate vaccine in order to prevent this disease an experimental model like this, rendering immunological reactions as revealed by the immunoperoxidase technique, would be useful.

Chagas' disease; Immunoperoxidase technique; Chronic myocarditis

La enfermedad de Chagas ha sido considerada como una de las causas más frecuentes de miocarditis crônica. Siendo descriptas Ias alteraciones inmunológicas, como patogenia para este tipo de enfermedad. Por tal motivo, se empleó la técnica de inmunoperoxidasa para la detección de depósitos de inmunoglobulinas en la miocardiopatía chagásica crônica experimental. Se utilizaron 41 ratones Swiss de 3 meses de vida, los mismos fueron inoculados intraperitonealmente con dosis entre 10 y 10(5); tripomastigotas de la cepa Tulahuen. La reinoculación se realizo 1 mes después con dosis entre 10² y 10(5); siendo sacrificados a los 6 (n=21) y 9 meses (n=9) de la primera inoculación, prévios estúdios elect roçar diográficos. Posteriormente se estudiaron los miocardios y músculos esqueléticos con técnicas histológicas de rutina, inmunoperoxidasa (método peroxidasa anti-peroxidasa) e inmunofluorescencia (directa e indirecta). Los métodos más sensibles para la detección de la enfermedad de Chagas crônica resultaron ser los estúdios histológicos (73%) y la electrocar-diografia (83%) a los 6 meses pi.y (89%) a los 9 meses p.i. (pos-infección). Se observaron diferentes alteraciones miocárdicas, desde infiltrados linfocitarios leves y focales en intersticio hasta reemplazo de miocitos por tejido conectivo. Los miocardio auriculares (21/23,91%) fueron más afectados que los ventrículos (9/23, 39%); mientras que las típicos quistes chagásicos resultaron excepcionales Los músculos esqueléticos (11/18 Y 7/9) presentaron distintos grados de lesión histológica, desdes leves a extensos infiltrados linfoplasmocitarios con presencia de fibras necróticas. Mientras que con la técnioa de inmunoperoxidasa, los antígenos se revelaron como depósitos granulares intracitoplasmáticos difusamente distribuidos, tanto para IgG como para Ig totales. La coincidência entre este método y Ias lesiones musculares histológicas fueron 11/18 (61%) a los 6 meses p.i.y 6/8 (75%) a los 9 meses p.i. Por otra parte, los depósitos de lg totales en corazón se observaron dispuestos difusamente, en forma finamente granular dentro de los miocitos ventriculares La coincidência entre ambas técnicas (inmunoperoxidasa e histología) resultó ser dei 36% y 66% para los animales sacrificados a los 6 y 9 meses p.i. respectivamente. Este fenômeno inmunológico se incremento notablemente con el curso crônico de la enfermedad. Con respecto a la sensibilidad, tanto la inmunoperoxidasa como la inmunofluorescencia directa fueron altamente sensibles en músculo esquelético (100%, p 0,01). Por otra parte, la técnica de inmunofluorescencia directa en corazón, evidencio pobres resultados, mientras que el método peroxidasa anti-peroxidasa incrementó su sensibilidad de 21,4% a los 6 meses pi. al 66.6% a los 9 meses p.i. (p 0.001). Este modelo experimental, en el cual se observan reacciones inmunológicas reveladas por la técnica de inmunoperoxidasa, podría resultar de utilidad, considerando la necesidad de obtener una vacuna adecuada para prevenir la miocardiopatía chagásica.

ORIGINAL ARTICLES

Immunoperoxidase technique in experimental chronic chagasic myocarditis

Técnica de inmunoperoxidasa en miocardiopatia chagásica crônica experimental

Maria Celina Morales, M.d.^I; José Milei, M.D.^II

^IResearch Fellow, National Academy of Medicine

^IIChief, Pathology Section. Hospital Institute of Cardiology. Assistant Professor of Internal Medicine, University of Buenos Aires

^{Address for reprints}

SUMMARY

Chagas'disease has been described as the commonest form of chronic myocarditis. An immunologic pathogenesis has been discribed for this form of the disease. So far, no immunoperoxidase technique has been used for the detection of immunological deposits in chronic experimental Chagas'myocardiopathy. Forty-one Swiss mice, three months old were inoculated intraperitoneally with doses between 10 and 10⁵ Tulahuen trypomastigotes. Mice were reinoculated one month after with doses between 10² and 10⁵ and sacrificed at 6 (n=21) and 9 months (n=9) after the first inoculation. ECGs were recorded before sacrifice. Immunoperoxidase technique (peroxidase-antiperoxidase method), immunofluorescence (direct and indirect) as well as histological studies were performed in myocardiums and skeletal muscles of the surviving animals. The most sensitive methods for detecting chronic chagasic infection were the routine histologic studies (73%) and the ECGs 83% and 89% on 6 and 9 mo. post-infected mice, respectively. Myocardial involvement varied from interstitial mild focal lymphocyte infiltrates up to replacement of myocytes by loose connective tissue. Atrial myocardiums (21/23, 91%) were more affected than ventricles (9/23, 39%). Typical chagasic nests were rarely found. Skeletal muscle involvement (11/18 and 7/9) varied from mild to extensive lymphocyte and plasmacell infiltrates, and necrotic fibers. The involved antigen were shown in skeletal muscles by the immunoperoxidase technique as diffusely arranged granular intracytoplasmatic deposit for both IgC and total immunoglobulins. The coincidence between this technique and histologic muscle lesions was 11/18 (61(%) in 6 mo. and 6/8 (75%) at 9 mo. post-infection. In heart, delicate granular deposits of total immunoglobulins were seen diffusely arranged within the ventricular myocytes; coincidence between immunoperoxidase technique anl histologic involvement increased from 36 to 66% in animals sacrifeced 6 and 9 mo. post-infection. This strongly stressed the increase of immunologic phenomena with the chronification of infection. Concerning sensitivity, immunoperoxidase and direct immunofluorescence were highly sensitive in skeletal muscle (100%, p < 0.01). Conversely, direct immunofluorescence technique showed poor results in heart while immunoperoxidase increased its sensitivity from 21.4% (at 6 mo.) to 66.6% (at 9 mo.) post-infection (p < 0.001). Considering the necessity of obtaining an adequate vaccine in order to prevent this disease an experimental model like this, rendering immunological reactions as revealed by the immunoperoxidase technique, would be useful.

Key words: Chagas' disease; Immunoperoxidase technique; Chronic myocarditis

RESUMEN

La enfermedad de Chagas ha sido considerada como una de las causas más frecuentes de miocarditis crônica. Siendo descriptas Ias alteraciones inmunológicas, como patogenia para este tipo de enfermedad.

Por tal motivo, se empleó la técnica de inmunoperoxidasa para la detección de depósitos de inmunoglobulinas en la miocardiopatía chagásica crônica experimental.

Se utilizaron 41 ratones Swiss de 3 meses de vida, los mismos fueron inoculados intraperitonealmente con dosis entre 10 y 10⁵; tripomastigotas de la cepa Tulahuen. La reinoculación se realizo 1 mes después con dosis entre 10² y 10⁵; siendo sacrificados a los 6 (n=21) y 9 meses (n=9) de la primera inoculación, prévios estúdios elect roçar diográficos.

Posteriormente se estudiaron los miocardios y músculos esqueléticos con técnicas histológicas de rutina, inmunoperoxidasa (método peroxidasa anti-peroxidasa) e inmunofluorescencia (directa e indirecta).

Los métodos más sensibles para la detección de la enfermedad de Chagas crônica resultaron ser los estúdios histológicos (73%) y la electrocar-diografia (83%) a los 6 meses pi.y (89%) a los 9 meses p.i. (pos-infección).

Se observaron diferentes alteraciones miocárdicas, desde infiltrados linfocitarios leves y focales en intersticio hasta reemplazo de miocitos por tejido conectivo.

Los miocardio auriculares (21/23,91%) fueron más afectados que los ventrículos (9/23, 39%); mientras que las típicos quistes chagásicos resultaron excepcionales

Los músculos esqueléticos (11/18 Y 7/9) presentaron distintos grados de lesión histológica, desdes leves a extensos infiltrados linfoplasmocitarios con presencia de fibras necróticas. Mientras que con la técnioa de inmunoperoxidasa, los antígenos se revelaron como depósitos granulares intracitoplasmáticos difusamente distribuidos, tanto para IgG como para Ig totales. La coincidência entre este método y Ias lesiones musculares histológicas fueron 11/18 (61%) a los 6 meses p.i.y 6/8 (75%) a los 9 meses p.i.

Por otra parte, los depósitos de lg totales en corazón se observaron dispuestos difusamente, en forma finamente granular dentro de los miocitos ventriculares La coincidência entre ambas técnicas (inmunoperoxidasa e histología) resultó ser dei 36% y 66% para los animales sacrificados a los 6 y 9 meses p.i. respectivamente.

Este fenômeno inmunológico se incremento notablemente con el curso crônico de la enfermedad. Con respecto a la sensibilidad, tanto la inmunoperoxidasa como la inmunofluorescencia directa fueron altamente sensibles en músculo esquelético (100%, p 0,01).

Por otra parte, la técnica de inmunofluorescencia directa en corazón, evidencio pobres resultados, mientras que el método peroxidasa anti-peroxidasa incrementó su sensibilidad de 21,4% a los 6 meses pi. al 66.6% a los 9 meses p.i. (p 0.001).

Este modelo experimental, en el cual se observan reacciones inmunológicas reveladas por la técnica de inmunoperoxidasa, podría resultar de utilidad, considerando la necesidad de obtener una vacuna adecuada para prevenir la miocardiopatía chagásica.

Texto completo disponível apenas em PDF.

Full text available only in PDF format.

ACKNOWLEDGEMENTS

Authors are indebted to Dr. P.M. Cossio and: his team for the fulfilment of immunofluorescent technique and to Dr. A. Molinolo for skilful assistance and advise in the attainment of immunoperoxidase technique. The authors also gratefully acknowledge the technical assistance of Noemi Borasit and the secretarial cooperation of Ana Becú.

Recebido para publicação em 25/7/1985.

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Address for reprints:

Dra. María Celina Morales.

Hospital Instituto de Cardiología.

Coronel Díaz 2423. 1425

Buenos Aires - Argentina.

Publication Dates

• Publication in this collection
  17 Feb 2011
• Date of issue
  Apr 1987

History

• Received
  25 July 1985