Natural killer cell activity in experimental paracoccidioidomycosis of the Syrian hamster

Peraçoli, Maria Terezinha Serrão; Fortes, Maria Rita Parise; Pereira da Silva, Maria Flávia; Montenegro, Mário Rubens

doi:10.1590/S0036-46651995000200007

Abstracts

The study evaluated the activity of NK cells during the course of experimental infection of hamsters with Paracoccidioides brasiliensis. Eigthy hamsters were infected with P. brasiliensis by intratesticular route and sacrificed at 24h, 48h, 96h, 1, 2, 4, 8 and 11 weeks of infection and compared to 40 noninfected hamsters employed as controls. These animals were submitted to the study of NK cytotoxic activity by a single-cell assay and humoral immune response by immunodiffusion and ELISA tests. The production of macrophage migration inhibitory factor in the presence of Phyto-hemagglutinin and P. brasiliensis antigen and histopathology of the lesions were evaluated at 1, 4, 8 and 11 weeks of infection. The infected animals displayed significantly high levels of NK activity during the four weeks of infection that decreased from the 8th week on when compared to controls. This impairment of NK activity was associated with depression of cell-mediated immune response and with increase in the extension of the histopathologic lesions. There was an inverse correlation between NK cell activity and specific antibody levels. The results suggest that after initial activation, NK cells were unable to control the fungus dissemination. The impairment of NK activity in the late stages of the infection might be related to immunoregulatory disturbances associated with paracoccidioidomycosis.

Paracoccidioidomycosis; Hamster; Cell-mediated immunity; NK cell activity; Antibodies

Com o objetivo de avaliar a atividade de células natural killer na paracoccidioidomicose experimental do hamster, 80 hamsters foram infectados por via intratesticular com Paracoccidioides brasiliensis e sacrificados após 24h, 48h, 96h, 1, 2, 4, 8 e 11 semanas de infecção. Como controle foram avaliados 40 hamsters normais, não infectados. Os animais foram submetidos ao estudo da atividade citotóxica de células NK pela técnica de "single-cell assay" e da resposta imune humoral pelas técnicas de imunodifusão dupla e Elisa. A produção do fator inibidor da migração de macrófagos em presença de Phytohemaglutinina e antígeno de P. brasiliensis e a histopatologia das lesões foram estudadas após 1,4, 8e 11 semanas de infecção. Os animais infectados, quando comparados aos controles, apresentaram níveis de atividade NK significativamente elevados durante as 4 primeiras semanas de infecção, havendo diminuição dessa atividade a partir da 8ª semana. Foi observada correlação inversa entre atividade NK e níveis de anticorpos específicos e, associação entre diminuição da atividade NK, depressão de resposta imune celular e aumento da extensão das lesões histopatológicas. Os resultados sugerem que após ativação inicial, as células NK são incapazes de controlar a disseminação do fungo pelos tecidos. A depressão da atividade NK na fase tardia da infecção parece estar relacionada com os distúrbios imunorregulatórios associados à paracoccidioidomicose.

IMMUNOLOGY

Natural killer cell activity in experimental paracoccidioidomycosis of the Syrian hamster

Atividade de células natural killer na paracoccidioidomicose experimental do hamster

Maria Terezinha Serrão Peraçoli^I; Maria Rita Parise Fortes^I; Maria Flávia Pereira da Silva^I; Mário Rubens Montenegro^II

^IDepartamento de Microbiologia e Imunologia, IB-UNESP, Botucatu-SP

^IIDepartamento de Patologia, Faculdade de Medicina de Botucatu, UNESP, Botucatu-SP

^{Correspondence to} Correspondence to: Dra. Maria Terezinha Serrão Peraçoli Departamento de Microbiologia e Imunologia, Instituto de Biociências, UNESP 18618-000 Botucatu, SP, Brazil

SUMMARY

The study evaluated the activity of NK cells during the course of experimental infection of hamsters with Paracoccidioides brasiliensis. Eigthy hamsters were infected with P. brasiliensis by intratesticular route and sacrificed at 24h, 48h, 96h, 1, 2, 4, 8 and 11 weeks of infection and compared to 40 noninfected hamsters employed as controls. These animals were submitted to the study of NK cytotoxic activity by a single-cell assay and humoral immune response by immunodiffusion and ELISA tests. The production of macrophage migration inhibitory factor in the presence of Phyto-hemagglutinin and P. brasiliensis antigen and histopathology of the lesions were evaluated at 1, 4, 8 and 11 weeks of infection. The infected animals displayed significantly high levels of NK activity during the four weeks of infection that decreased from the 8^th week on when compared to controls. This impairment of NK activity was associated with depression of cell-mediated immune response and with increase in the extension of the histopathologic lesions. There was an inverse correlation between NK cell activity and specific antibody levels. The results suggest that after initial activation, NK cells were unable to control the fungus dissemination. The impairment of NK activity in the late stages of the infection might be related to immunoregulatory disturbances associated with paracoccidioidomycosis.

Keywords: Paracoccidioidomycosis; Hamster; Cell-mediated immunity; NK cell activity; Antibodies.

RESUMO

Com o objetivo de avaliar a atividade de células natural killer na paracoccidioidomicose experimental do hamster, 80 hamsters foram infectados por via intratesticular com Paracoccidioides brasiliensis e sacrificados após 24h, 48h, 96h, 1, 2, 4, 8 e 11 semanas de infecção. Como controle foram avaliados 40 hamsters normais, não infectados. Os animais foram submetidos ao estudo da atividade citotóxica de células NK pela técnica de "single-cell assay" e da resposta imune humoral pelas técnicas de imunodifusão dupla e Elisa. A produção do fator inibidor da migração de macrófagos em presença de Phytohemaglutinina e antígeno de P. brasiliensis e a histopatologia das lesões foram estudadas após 1,4, 8e 11 semanas de infecção. Os animais infectados, quando comparados aos controles, apresentaram níveis de atividade NK significativamente elevados durante as 4 primeiras semanas de infecção, havendo diminuição dessa atividade a partir da 8ª semana. Foi observada correlação inversa entre atividade NK e níveis de anticorpos específicos e, associação entre diminuição da atividade NK, depressão de resposta imune celular e aumento da extensão das lesões histopatológicas. Os resultados sugerem que após ativação inicial, as células NK são incapazes de controlar a disseminação do fungo pelos tecidos. A depressão da atividade NK na fase tardia da infecção parece estar relacionada com os distúrbios imunorregulatórios associados à paracoccidioidomicose.

Full text available only in PDF format.

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ACKNOWLEDGEMENTS

The authors are grateful to Mr. Antonio Emilio de Oliveira, Mrs. Maria Antonia M. da Palma and Mr. Luis Alquati for technical assistance and to Dr. Paulo Curi for the statistical analysis. This study was supported by FUNDUNESP, grant 082/92 DFP.

Recebido para publicação em 23/08/1994

Aceito para publicação em 21/02/1995

References

1. BAVA, J.A.; MISTCHENKO, A.S.; PALACIOS, M.F. et al. - Lymphocyte subpopulations and cytokine production in paracoccidioidomycosis patients. Microbiol. Immunol., 35: 167-174, 1991.
2. BIAGIONI, L.; SOUZA, M.J.; CHAMMA, L.G. et al. - Serology of paracoccidioidomycosis. II. Correlation between class-specific antibodies and clinical forms of the disease. Trans. roy. Soc. trop. Med. Hyg., 78: 617-621, 1984.
3. BISTONI, F.; BACCARINI, M.; BLASI, E. et al. - Correlation between "in vivo" and "in vitro" studies of modulation resistance of experimental Candida albicans infection by cyclophosphamide in mice. Infect. Immun., 40: 46-55, 1983.
4. CALICH, V.L.G.; PURCHIO, A. & PAULA, C.R. - A new fluorescent viability test for fungi cells. Mycopathologia (Den Haag), 66: 175-177, 1978.
5. CHEQUER-BOU-HABIB, D.C.; OLIVEIRA-NETTO, M.P.; FERREIRA-DA-CRUZ, M.T. & GALVĂO-CASTRO, B. - The possible role of circulating immunecomplexes in the deficiency of cell-mediated immunity in paracoccidioidomycosis. Braz. J. med. biol. Res., 22: 205-212, 1989.
6. COELHO, K.I.R.; DEFAVERI, J.; REZKALLAH-IWASSO, M.T. & PERAÇOLI, M.T.S. - Experimental paracoccidioidomycosis. In: FRANCO, M.; LACAZ, C.S.; RESTREPO-MORENO, A. & DEL NEGRO, G. ed. Paracoccidioidomycosis. Boca Raton, CRC Press, 1994. p. 87-107.
7. CONVERSE, P.J. & BJUNE, G. - Natural killer (NK) cell activity and reversal reactions in leprosy. Int. J. Leprosy, 54: 503-509, 1986.
8. COSTA, J.C.; PAGANO, P.M.G.; BECHELLI, L.M.; FIORILLO, A.M. & FILHO, E.C.L. - Lymphocyte transformation test in patients with paracoccidioidomycosis. Mycopathologia (Den Haag), 84: 55-63, 1983.
9. FAVA NETTO, C.; VEGAS, V.S.; SCIANNAMEA, I.M. & GUARNIERI, D.B. - Antígeno polissacarídico do Paracoccidioides brasiliensis. Estudo do tempo de cultivo do P. brasiliensis necessário ao preparo do antígeno. Rev. Inst. Med. trop. S. Paulo, 11: 177-181, 1969.
10. FELDMEIER, H.; GASTL, G.A.; POGGENSEE, V. et al. - Relationship between intensity of infection and immunomodulation in human schistosomiasis. II. NK cell activity and "in vitro" lymphocyte proliferation. Clin. exp. Immunol., 60: 234-240, 1985.
11. FRANCO, M.; PERAÇOLI, M.T.S.; SOARES, A.M.V.C. et al. - Host-parasite relationship in paracoccidioidomycosis. Curr. Top. med. Mycol., 5: 115-149, 1993.
12. GRANGER, D.C. & HOBBS, M.M. - Do natural killer cells help protect us from disease caused by pathogenic fungi? Amer. Rev. resp. Dis., 138: 510-511, 1988.
13. HATCHER, F.M. & KUHN, P.E. - Destruction of Trypanosoma cruzi by natural killer cells. Science, 218: 295-296, 1982.
14. HIDORE, M.R. & MURPHY, J.W. - Correlation of natural killer cell activity and clearance of Cryptococcus neoformans from mice after adoptive transfer of splenic nylon wool nonadherent cells. Infect. Immun., 51: 547-555, 1986.
15. HIDORE, M.R.; NAVABI, N.; SONLEITNER, F. & MURPHY, J.W. - Murine natural killer cells rare fungicidal to Cryptococcus neoformans. Infect. Immun., 59: 1747-1754, 1991.
16. JIMENEZ, B. & MURPHY, J.W. - "In vitro" effects of natural killer cells against Paracoccidioides brasiliensis yeast phase. Infect. Immun., 46: 552-558, 1984.
17. LOWRY, O.H.; ROSEBROUGH, N.J.; FARR, A.L. & RANDALL, R.J. - Protein measurement with the Folin phenol reagent. J. biol. Chem., 193: 265-275, 1951.
18. MENDES-GIANNINI, M.J.S.; CAMARGO, M.E.; LACAZ, C.S. & FERREIRA, A.W. - Immunoenzymatic absorption test for serodiagnosis of paracoccidioidomycosis. J. clin. Microbiol., 20: 103-108, 1984.
19. MOTA, N.G.S.; REZKALLAH-IWASSO, M.T.; PERAÇOLI, M.T.S. et al. - Correlation between cell-mediated immunity and clinical forms of paracoccidioidomycosis. Trans. roy. Soc. trop. Med. Hyg., 79: 765-772, 1985.
20. MUSATTI, C.C.; REZKALLAH-IWASSO, T.; MENDES, E. & MENDES, N.F. - "In vivo" and "in vitro" evaluation of cell-mediated immunity in patients with paracoccidioidomycosis. Cell Immunol., 24: 365-378, 1976.
21. NAIFF, R.D.; FERREIRA, L.C.L.; BARRET, T.V.; NAIFF, M.F. & ARIAS, J.R. - Paracoccidioidomicose enzoótica em tatus (Dasypus novemcinctus) no estado do Pará. Rev. Inst. Med. trop. S. Paulo, 28: 19-27, 1986.
22. PEDERSEN, B.K.; THONSEN, B.S. & NIELSEN, H. - Inhibition of natural killer cell activity by antigen-antibody complexes. Allergy, 41: 568-574, 1986.
23. PERAÇOLI, M.T.S.; MOTA, N.G.S. & MONTENEGRO, M.R. - Experimental paracoccidioidomycosis in the Syrian hamster: morphology and correlation of lesions with humoral and cell-mediated immunity. Mycopathologia (Den Haag), 79: 7-17, 1982.
24. PERAÇOLI, M.T.S.; SOARES, A.V.C.; MENDES, R.P. et al. - Studies of natural killer cells in patients with paracoccidioidomycosis. J. Med. vet. Mycol., 29: 373-380, 1991.
25. PUCCIA, R.; SCHENKMAN, S.; GORIN, P.A. & TRAVASSOS, L.R. - Exocellular components of Paracoccidioitles brasiliensis: identification of a specific antigen. Infect. Immun., 53: 199-206, 1986.
26. REYNOLDS, C.W.; TIMMONEN, T. & HERBERMAN, R.B. - NK activity in the rat. I. Isolation and characterization of the effector cells. J. Immunol., 127: 282-287, 1981.
27. REZKALLAH-IWASSO, M.T.; SOARES, A.M.V.C.; SANTANA, J. et al. - Interleukin-2 in paracoccidioidomycosis. Rev. argent. Micol., 15: 59, 1992.
28. TANI, E.M.; FRANCO, M.F.; PERAÇOLI, M.T.S. & MONTENEGRO, M.R. - Experimental pulmonary paracoccidioidomycosis in the Syrian hamster: morphology and correlation of lesions with immune response. J. Med. vet. Mycol., 25: 291-300, 1987.
29. TEALE, D.M.; REES, R.C.; CLARK, A. & POTTER, C.W. - Detection and characterization of natural killer cells in Syrian golden hamsters. Europ. J. Cancer clin. Oncol., 4: 537-545, 1983.
30. TRINCHIERI, G. - Biology of natural killer cells. Advanc. Immunol., 47: 187-375, 1989.
31. WAHLIN, B.; ALSHEIKHLY, A.; PERLMANN, P.; SCHREIBER, R.D. & MULLER-EBERHARD, H.J. - Enumeration and characterization of human natural killer cells by a modified single cell assay. Scand. J. Immunol., 19: 529-539, 1984.
32. WERKMEISTER, J.A.; RODER, J.C.; CURRY, C. & PROSS, H. - The effect of unphosphorylated and phosphorylated sugar moieties in human and mouse natural killer cell activity: is there selective inhibition at the level of target recognition and lytic acceptor site? Cell. Immunol., 80: 172-186, 1983.
33. WHITESIDE, L.T. & HERBERMAN, R.B. - Short analytical review the role of natural killer cells in human disease. Clin. Immunol. Immunopath., 53: 1-9, 1989.
34. WILLIAMS, M.L.; LOUGHRAN Jr., T.P.; KIDD, P.G. & STARKEBAUM, G.A. - Polyclonal proliferation of activated suppresor/cytotoxic T cells with transient depression of natural killer cell function in acute infectious mononucleosis. Clin. exp. Immunol., 77: 71-76, 1989.
35. ZAR, J.H. - Two factors analysis of variance. In: ZAR, J. H. Biostatistical analysis. 2. ed. New Jersey, Prentice Hall, 1984. p. 206-234.

Correspondence to:

Dra. Maria Terezinha Serrão Peraçoli

Departamento de Microbiologia e Imunologia, Instituto de Biociências, UNESP

18618-000 Botucatu, SP, Brazil

Publication Dates

Publication in this collection
22 Feb 2007
Date of issue
Apr 1995

History

Accepted
21 Feb 1995
Received
23 Aug 1994

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. BAVA, J.A.; MISTCHENKO, A.S.; PALACIOS, M.F. et al. - Lymphocyte subpopulations and cytokine production in paracoccidioidomycosis patients. Microbiol. Immunol., 35: 167-174, 1991.

[2] 2. BIAGIONI, L.; SOUZA, M.J.; CHAMMA, L.G. et al. - Serology of paracoccidioidomycosis. II. Correlation between class-specific antibodies and clinical forms of the disease. Trans. roy. Soc. trop. Med. Hyg., 78: 617-621, 1984.

[3] 3. BISTONI, F.; BACCARINI, M.; BLASI, E. et al. - Correlation between "in vivo" and "in vitro" studies of modulation resistance of experimental Candida albicans infection by cyclophosphamide in mice. Infect. Immun., 40: 46-55, 1983.

[4] 4. CALICH, V.L.G.; PURCHIO, A. & PAULA, C.R. - A new fluorescent viability test for fungi cells. Mycopathologia (Den Haag), 66: 175-177, 1978.

[5] 5. CHEQUER-BOU-HABIB, D.C.; OLIVEIRA-NETTO, M.P.; FERREIRA-DA-CRUZ, M.T. & GALVĂO-CASTRO, B. - The possible role of circulating immunecomplexes in the deficiency of cell-mediated immunity in paracoccidioidomycosis. Braz. J. med. biol. Res., 22: 205-212, 1989.

[6] 6. COELHO, K.I.R.; DEFAVERI, J.; REZKALLAH-IWASSO, M.T. & PERAÇOLI, M.T.S. - Experimental paracoccidioidomycosis. In: FRANCO, M.; LACAZ, C.S.; RESTREPO-MORENO, A. & DEL NEGRO, G. ed. Paracoccidioidomycosis. Boca Raton, CRC Press, 1994. p. 87-107.

[7] 7. CONVERSE, P.J. & BJUNE, G. - Natural killer (NK) cell activity and reversal reactions in leprosy. Int. J. Leprosy, 54: 503-509, 1986.

[8] 8. COSTA, J.C.; PAGANO, P.M.G.; BECHELLI, L.M.; FIORILLO, A.M. & FILHO, E.C.L. - Lymphocyte transformation test in patients with paracoccidioidomycosis. Mycopathologia (Den Haag), 84: 55-63, 1983.

[9] 9. FAVA NETTO, C.; VEGAS, V.S.; SCIANNAMEA, I.M. & GUARNIERI, D.B. - Antígeno polissacarídico do Paracoccidioides brasiliensis. Estudo do tempo de cultivo do P. brasiliensis necessário ao preparo do antígeno. Rev. Inst. Med. trop. S. Paulo, 11: 177-181, 1969.

[10] 10. FELDMEIER, H.; GASTL, G.A.; POGGENSEE, V. et al. - Relationship between intensity of infection and immunomodulation in human schistosomiasis. II. NK cell activity and "in vitro" lymphocyte proliferation. Clin. exp. Immunol., 60: 234-240, 1985.

[11] 11. FRANCO, M.; PERAÇOLI, M.T.S.; SOARES, A.M.V.C. et al. - Host-parasite relationship in paracoccidioidomycosis. Curr. Top. med. Mycol., 5: 115-149, 1993.

[12] 12. GRANGER, D.C. & HOBBS, M.M. - Do natural killer cells help protect us from disease caused by pathogenic fungi? Amer. Rev. resp. Dis., 138: 510-511, 1988.

[13] 13. HATCHER, F.M. & KUHN, P.E. - Destruction of Trypanosoma cruzi by natural killer cells. Science, 218: 295-296, 1982.

[14] 14. HIDORE, M.R. & MURPHY, J.W. - Correlation of natural killer cell activity and clearance of Cryptococcus neoformans from mice after adoptive transfer of splenic nylon wool nonadherent cells. Infect. Immun., 51: 547-555, 1986.

[15] 15. HIDORE, M.R.; NAVABI, N.; SONLEITNER, F. & MURPHY, J.W. - Murine natural killer cells rare fungicidal to Cryptococcus neoformans. Infect. Immun., 59: 1747-1754, 1991.

[16] 16. JIMENEZ, B. & MURPHY, J.W. - "In vitro" effects of natural killer cells against Paracoccidioides brasiliensis yeast phase. Infect. Immun., 46: 552-558, 1984.

[17] 17. LOWRY, O.H.; ROSEBROUGH, N.J.; FARR, A.L. & RANDALL, R.J. - Protein measurement with the Folin phenol reagent. J. biol. Chem., 193: 265-275, 1951.

[18] 18. MENDES-GIANNINI, M.J.S.; CAMARGO, M.E.; LACAZ, C.S. & FERREIRA, A.W. - Immunoenzymatic absorption test for serodiagnosis of paracoccidioidomycosis. J. clin. Microbiol., 20: 103-108, 1984.

[19] 19. MOTA, N.G.S.; REZKALLAH-IWASSO, M.T.; PERAÇOLI, M.T.S. et al. - Correlation between cell-mediated immunity and clinical forms of paracoccidioidomycosis. Trans. roy. Soc. trop. Med. Hyg., 79: 765-772, 1985.

[20] 20. MUSATTI, C.C.; REZKALLAH-IWASSO, T.; MENDES, E. & MENDES, N.F. - "In vivo" and "in vitro" evaluation of cell-mediated immunity in patients with paracoccidioidomycosis. Cell Immunol., 24: 365-378, 1976.

[21] 21. NAIFF, R.D.; FERREIRA, L.C.L.; BARRET, T.V.; NAIFF, M.F. & ARIAS, J.R. - Paracoccidioidomicose enzoótica em tatus (Dasypus novemcinctus) no estado do Pará. Rev. Inst. Med. trop. S. Paulo, 28: 19-27, 1986.

[22] 22. PEDERSEN, B.K.; THONSEN, B.S. & NIELSEN, H. - Inhibition of natural killer cell activity by antigen-antibody complexes. Allergy, 41: 568-574, 1986.

[23] 23. PERAÇOLI, M.T.S.; MOTA, N.G.S. & MONTENEGRO, M.R. - Experimental paracoccidioidomycosis in the Syrian hamster: morphology and correlation of lesions with humoral and cell-mediated immunity. Mycopathologia (Den Haag), 79: 7-17, 1982.

[24] 24. PERAÇOLI, M.T.S.; SOARES, A.V.C.; MENDES, R.P. et al. - Studies of natural killer cells in patients with paracoccidioidomycosis. J. Med. vet. Mycol., 29: 373-380, 1991.

[25] 25. PUCCIA, R.; SCHENKMAN, S.; GORIN, P.A. & TRAVASSOS, L.R. - Exocellular components of Paracoccidioitles brasiliensis: identification of a specific antigen. Infect. Immun., 53: 199-206, 1986.

[26] 26. REYNOLDS, C.W.; TIMMONEN, T. & HERBERMAN, R.B. - NK activity in the rat. I. Isolation and characterization of the effector cells. J. Immunol., 127: 282-287, 1981.

[27] 27. REZKALLAH-IWASSO, M.T.; SOARES, A.M.V.C.; SANTANA, J. et al. - Interleukin-2 in paracoccidioidomycosis. Rev. argent. Micol., 15: 59, 1992.

[28] 28. TANI, E.M.; FRANCO, M.F.; PERAÇOLI, M.T.S. & MONTENEGRO, M.R. - Experimental pulmonary paracoccidioidomycosis in the Syrian hamster: morphology and correlation of lesions with immune response. J. Med. vet. Mycol., 25: 291-300, 1987.

[29] 29. TEALE, D.M.; REES, R.C.; CLARK, A. & POTTER, C.W. - Detection and characterization of natural killer cells in Syrian golden hamsters. Europ. J. Cancer clin. Oncol., 4: 537-545, 1983.

[30] 30. TRINCHIERI, G. - Biology of natural killer cells. Advanc. Immunol., 47: 187-375, 1989.

[31] 31. WAHLIN, B.; ALSHEIKHLY, A.; PERLMANN, P.; SCHREIBER, R.D. & MULLER-EBERHARD, H.J. - Enumeration and characterization of human natural killer cells by a modified single cell assay. Scand. J. Immunol., 19: 529-539, 1984.

[32] 32. WERKMEISTER, J.A.; RODER, J.C.; CURRY, C. & PROSS, H. - The effect of unphosphorylated and phosphorylated sugar moieties in human and mouse natural killer cell activity: is there selective inhibition at the level of target recognition and lytic acceptor site? Cell. Immunol., 80: 172-186, 1983.

[33] 33. WHITESIDE, L.T. & HERBERMAN, R.B. - Short analytical review the role of natural killer cells in human disease. Clin. Immunol. Immunopath., 53: 1-9, 1989.

[34] 34. WILLIAMS, M.L.; LOUGHRAN Jr., T.P.; KIDD, P.G. & STARKEBAUM, G.A. - Polyclonal proliferation of activated suppresor/cytotoxic T cells with transient depression of natural killer cell function in acute infectious mononucleosis. Clin. exp. Immunol., 77: 71-76, 1989.

[35] 35. ZAR, J.H. - Two factors analysis of variance. In: ZAR, J. H. Biostatistical analysis. 2. ed. New Jersey, Prentice Hall, 1984. p. 206-234.