Paracoccidioidomycosis: a sequential histopathologic study of lesions in experimentally-infected rats

Kerr, I.B.; Araripe, J.R.; Oliveira, P.C.; Lenzi, H.L.

doi:10.1590/S0036-46651988000500003

Abstracts

Female albino rats were used for the sequential histopathological study of experimental paracoccidioidomycosis. The animals were inoculated intraperitoneally with a strain of Paracoccidioides brasiliensis in the yeast-like phase, and sacrificed at given intervals from 1 to 168 days after inoculation; each animal received an inoculum of 4 x 10(6) cells in 0.8 ml of saline. The control group received saline containing scrapings of the culture medium. Tissue from the inoculation site was examined. The cellular population, the extracellular matrix, and the presence and characteristics of fungi were analysed in the inflammatory granulomatous process by light microscopy. The results allowed to separate the kinetic of the inflammatory response into three stages: 1) neutrophilic or macrophagic-neutrophilic; 2) pre-granulomatous; 3) granulomatous. Synthesis of the extracellular matrix began with the depositing of fibrin-like material, and increased gradually with deposits of collagen, proteoglycans, and glycoproteins. Parasites were present in all of the examined periods. Recurrences of the disease were clearly shown through the concurrence of recently-formed granulomas with older granulomas, implying that this type of granulomatous process does not eliminate the disease, nor is it able to limit fungal dissemination over a prolonged period of time.

Paracoccidioidomycosis; Paracoccidioides brasiliensis; Granuloma; Extracellular matrix; Collagen; Proteoglycans

Foram utilizados ratos albinos, fêmeas, para o estudo histopatológico sequencial da paracoccidioidomicose experimental. Os animais39 foram inoculados intraperitonealmente com uma cepa de Paracoccidioides brasiliensis na fase leveduriforme e sacrificados, em determinados intervalos, a partir de 1 a 168 dias pós-infecção; cada animal recebeu um inóculo de 4 x 10(6) células em 0,8 ml de salina. Os animais controles receberam salina contendo raspado do meio de cultura. Foram estudados tecidos correspondentesà área de inoculação. Analisou-se pela microscopia óptica o processo inflamatório granulomatoso em todo o seu conjunto, estudando a população celular, a matriz extracelular e a presença e características do fungo. Os resultados possibilitaram desmembrar a cinética da resposta inflamatória em três fases: 1) neutrofílica ou macrofágica-neutrofílica; 2) pré-granulomatosa; 3) granulomatosa. A síntese de matriz extracelular iniciou-se pela deposição de material fibrinóide, intensificando-se de modo gradativo com depósito de colágeno, de proteoglicanos e glicoproteínas. Os parasitos estavam presentes em todas as fases estudadas. Períodos de reativação da doença eram nitidamente evidenciados através da concomitância de granulomas recém-formados com granulomas mais antigos, indicando que o processo granulomatoso neste modelo não resolve a doença, nem tão pouco consegue limitar a disseminação do fungo por um período prolongado.

ORIGINAL ARTICLES

Paracoccidioidomycosis: a sequential histopathologic study of lesions in experimentally-infected rats

Paracoccidioidomicose: estudo histopatológico sequencial das lesões em ratos experimentalmente infectados

I.B. Kerr^I; J.R. Araripe^{I, III}; P.C. Oliveira^II; H.L. Lenzi^I

^IDepartment of Pathology of the Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ., Brazil

^IIDepartment of Mycology of the Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ., Brazil

^IIIRecipient of a CNPq Fellowship (Nº 822868-86.5)

^{Address for correspondence} Address for correspondence: Itália B. Kerr Departamento de Patologia Instituto Oswaldo Cruz, FIOCRUZ Caixa Postal 926. CEP 20010 Rio de Janeiro, RJ, Brasil

SUMMARY

Female albino rats were used for the sequential histopathological study of experimental paracoccidioidomycosis. The animals were inoculated intraperitoneally with a strain of Paracoccidioides brasiliensis in the yeast-like phase, and sacrificed at given intervals from 1 to 168 days after inoculation; each animal received an inoculum of 4 x 10⁶ cells in 0.8 ml of saline. The control group received saline containing scrapings of the culture medium.

Tissue from the inoculation site was examined. The cellular population, the extracellular matrix, and the presence and characteristics of fungi were analysed in the inflammatory granulomatous process by light microscopy.

The results allowed to separate the kinetic of the inflammatory response into three stages: 1) neutrophilic or macrophagic-neutrophilic; 2) pre-granulomatous; 3) granulomatous.

Synthesis of the extracellular matrix began with the depositing of fibrin-like material, and increased gradually with deposits of collagen, proteoglycans, and glycoproteins. Parasites were present in all of the examined periods.

Recurrences of the disease were clearly shown through the concurrence of recently-formed granulomas with older granulomas, implying that this type of granulomatous process does not eliminate the disease, nor is it able to limit fungal dissemination over a prolonged period of time.

Key words: Paracoccidioidomycosis; Paracoccidioides brasiliensis; Granuloma; Extracellular matrix; Collagen; Proteoglycans.

RESUMO

Foram utilizados ratos albinos, fêmeas, para o estudo histopatológico sequencial da paracoccidioidomicose experimental. Os animais³⁹ foram inoculados intraperitonealmente com uma cepa de Paracoccidioides brasiliensis na fase leveduriforme e sacrificados, em determinados intervalos, a partir de 1 a 168 dias pós-infecção; cada animal recebeu um inóculo de 4 x 10⁶ células em 0,8 ml de salina. Os animais controles receberam salina contendo raspado do meio de cultura.

Foram estudados tecidos correspondentesà área de inoculação. Analisou-se pela microscopia óptica o processo inflamatório granulomatoso em todo o seu conjunto, estudando a população celular, a matriz extracelular e a presença e características do fungo.

Os resultados possibilitaram desmembrar a cinética da resposta inflamatória em três fases: 1) neutrofílica ou macrofágica-neutrofílica; 2) pré-granulomatosa; 3) granulomatosa.

A síntese de matriz extracelular iniciou-se pela deposição de material fibrinóide, intensificando-se de modo gradativo com depósito de colágeno, de proteoglicanos e glicoproteínas. Os parasitos estavam presentes em todas as fases estudadas.

Períodos de reativação da doença eram nitidamente evidenciados através da concomitância de granulomas recém-formados com granulomas mais antigos, indicando que o processo granulomatoso neste modelo não resolve a doença, nem tão pouco consegue limitar a disseminação do fungo por um período prolongado.

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Recebido para publicação em 10/3/1988.

1. BEELEN, R.H.J.; FLUITSMA, D.M. & HOEFSMIT, E.C.M. - The cellular composition of omentum milky spots and the ultrastructure of milky spot macrophages and reticulum cells. J. reticuloendoth. Soc, 28: 585-599, 1980.
2. BEER, D.J.; MATLOFF, S.M. & ROCKLIN, R.E. - The influence of histamine on immune and inflammatory response. Advanc. Immunol., 35: 209-268, 1984.
3. BIAGIONI, L.M.V.; ORSI, S.; CHAMMA, L.G.; SADATSUNE, T. & FRANCO, M. - Imunoglobulinas e C₃ no granuloma paracoccidióidico. Rev. Inst. Med. trop. S. Paulo, 29: 97-103, 1987.
4. BIAGIONI, L.; SOUZA, M.J.; CHAMMA, L.G.; MENDES, R.P.; MARQUES, S.A.; MOTA, N.G.S. & FRANCO, M. - 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.
5. BJERMER, L.; ENGSTRÖM-LAURENT, A.; THUNELL, M. & HÄLLGREN, R. - The mast cell and signs of pulmonary fibroblast activation in sarcoidosis. Int. Arch. Allergy, 82: 298-301, 1987.
6. BRITO, T. & FAVA NETTO, C. - Disseminated experimental South American blastomycosis of the Guinea pig; a pathologic and immunologic study. Path. et Microbiol. (Basel), 26: 29-43, 1963.
7. BURGER, D.R. & VETTO, R.M. - Hypothesis. Vascular endothelium as a major participant in T-lymphocyte immunity. Cell. Immunol., 70: 357-361, 1982.
8. CALICH, V.L.G.; KIPNIS,T.L.; MARIANO, M.; FAVA-NETO, C. & DIAS DA SILVA, W. - The activation of the complement system by Paracoccidioides brasiliensis in vitro: its opsonic effect and possible signiticance for an in vivo model of infection. Clin. Immunol. Immunopath., 12: 20-30, 1979.
9. CALICH, V.L.G.; VAZ, C.A.C. & BURGER, E. - PMN chemotactic factor produced by glass-adherent cells in the acute inflammation caused by Paracoccidioides brasiliensis Brit. J. exp. Path., 66: 57-65, 1985.
10. CARSON, F.L.; MARTIN, J.H. & LYNN, J.A.- Formalin fixation for electron microscopy. A reevaluation. Amer. J. clin. Path., 59: 365-373,1973.
11. CARVALHAES, M.S.; SILVA, W.D.; BIRMAN, E.G.; SANT'ANNA, O.A.; ABRAHAMSOHN, P. & LIBERMAN KIPNIS, T. - Experimental paracoccidioidomycosis in high and low antibody-producer mice. 1. Evolution of the disease, us correlation with the humoral immune response and the patterns of tissue lesions. Ann. Immunol. (Paris), Serie C, 137C: 127-141, 1986.
12. CLAMAN, H.N. - Mast cell, T cells and abnormal fibrosis, Immunol. Today, 6: 192-196, 1985.
13. DEXTER, T.M.; STODDART, R.W. & QUAZZAZ, S.T.A. - What are mast cells for? Nature, 291: 110-111, 1981.
14. DUNN, M.A. - Liver fibrosis in schistosomiasis. In: SLUTZKY, G.M., ed. - The Biochemistry of parasites. New York, Pergamon Press, 1980. p. 191-199.
15. FREUNDLICH, B.; BOMALASKI, J.S.; NEILSON, E. & JIMENEZ, S.A. - Regulation of fibroblast proliferation and collagen synthesis by cytokines. Immunol. Today, 7: 303-307, 1986.
16. GOIHMAN-YAHR, M.; ESSENFELD-YAHR. E.; ALBORNOZ, M.C.; YARZÁBAL, L.; GÓMEZ, M.H.; SAN MARTIN, B.; OCANTO, A.; GIL, F. & CONVIT, J. - Defect of in vitro digestive ability of polymorphonuclear leukocytes in paracoccidioidomycosis. Infect. Immun., 28: 557-566, 1980.
17. IABUKI, K. & MONTENEGRO, MR. - Experimental paracoccidioidomycosis in the Syrian hamster: Morphology, ultrastructure and correlation of lesions with presence of specific antigens and serum levels of antibodies. Mycopathologia (Deo Haag), 67: 131-141, 1979.
18. JONES, P.A. & WERB, Z. - Degradation of connective tissue matrices by macrophages. II influence of matrix composition on proteolysis of glycoproteins, elastin, and collagen by macrophages in culture. J. exp. Med., 152: 1527-1536, 1980.
19. JUNQUEIRA, L.C.U.; BIGNOLAS, G. & BRENTANI, R.R. - Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem. J., 11: 447-455, 1979.
20. JUNQUEIRA, L.C.U. & MONTES, G.S. - Biology of collagen-proteoglycan interaction. Arch, histol. jap., 46: 589-629, 1983.
21. KERR. I.B.; OLIVEIRA, P.C. & SCHAEFFER, G.V. - Influence of inoculum size on the development of intraperitoneal paracoccidioidomycosis in rats. J. infect. Dis., 149: 821, 1984.
22. KERR, I.B. SCHAEFFER, G.V. & MIRANDA, D.S. - Sex hormones and susceptibility of the rat to paracoccidioidomycosis. Mycopathologia (Den Haag), 88: 149-154, 1984.
23. KUETTNER, K.E. & KIMURA, J.H. - Proteoglycans an overview. J. cell. Biochem., 27: 327-336, 1985.
24. LENNERT, K. - Malignant lymphomas other than Hodgkin's disease. Berlin, Spring-Verlag, 1978. p.
25. LINARES, L.I. & FRIEDMAN, L. - Experimental paracoccidioidomycosis in mice. Infect. Immun., 5: 681-687,1972.
26. MACKINNON, J.E. - Pathogenesis of South American Blastomycosis. Trans. roy. Soc. trop. Med. Hyg., 53: 487-494, 1959.
27. McEWEN, J.G.; BRUMMER, E.; STEVENS, D.A. & RESTREPO, A. - Effect of murine polymorphonuclear leukocytes on the yeast form of Paracoccidioides brasiliensis Amer. J. trop. Med. Hyg., 36: 603-608, 1987.
28. McEWEN, J.G.; SUGAR, A.M.; BRUMMER, E., RESTREPO, A. & STEVENS, D.A. - Toxic effect of products of oxidative metabolism on the yeast form of Paracoccidioides brasiliensis J. med. Microbiol., 18: 423-428, 1984.
29. MOSCARDI, M. & FRANCO, M.F. - Paracoccidioidomicose experimental do camundongo. II- Infecção intraperitoneal após sensibilização prévia. Rev. Inst. Med. trop. S. Paulo, 23: 204-211, 1981.
30. 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.
31. POSTLETHWAITE, A.E.; JACKSON, B.K.; BEACHEY, E.H. & KANG, A.H. - Formation of multinucleated giant cells from human monocyte percursors. J. exp. Med., 155: 168-178, 1982.
32. RESTREPO, A. & VÉLEZ, H. - Efectos de la fagocitosis in vitro sobre el Paracoccidioides brasiliensis Sabouraudia, 13(part 1): 10-21, 1975.
33. SCHWARTZ, L.B. & AUSTEN, K.F. - Structure and function of the chemical mediators of mast cells. Progr. Allergy, 34: 271-321, 1984.
34. TEIXEIRA, G.A.; MACHADO FILHO, J. & MIRANDA, J.L. - Blastomicose sul-americana experimental. Estudo experimental em ratos com considerate relativas à patogenia das lesões. Hospital (Rio de J.), 68: 1081-1096, 1965.
35. TURK, J.L. & NARAYAMA, R.B. - The origin, morphology, and function of epithelioid cells. Immunobiology, 161:274-282, 1982.
36. WAHL, S.M. & WAHL, L.M. - Modulation of fibroblast growth and function by monokines and lymphokines. Lymphokine Res., 2: 179-201, 1981.
37. WEINBERG, J.B.; HOBBS, M.M. & MISUKONIS, M.A. - Recombinant human-interferon induces human monocyte polykaryon formation. Proc. nat. Acad. Sci. (Wash.), 81: 4554-4557, 1984.
38. WHITAKER, D. & PAPADIM1TRIOU, J. - Mesothelial healing: morphological and kinetic investigations. J. Path., 145:159-175, 1985.
39. WOLMAN, M. & KASTEN, F.H. - Polarized light microscopy in the study of the molecular structure of collagen and reticulin. Histochemistry, 85:41-49, 1986.

Address for correspondence:

Itália B. Kerr

Departamento de Patologia

Instituto Oswaldo Cruz, FIOCRUZ

Caixa Postal 926. CEP 20010 Rio de Janeiro, RJ, Brasil

Publication Dates

Publication in this collection
17 Feb 2011
Date of issue
Oct 1988

History

Received
10 Mar 1988

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

[1] 1. BEELEN, R.H.J.; FLUITSMA, D.M. & HOEFSMIT, E.C.M. - The cellular composition of omentum milky spots and the ultrastructure of milky spot macrophages and reticulum cells. J. reticuloendoth. Soc, 28: 585-599, 1980.

[2] 2. BEER, D.J.; MATLOFF, S.M. & ROCKLIN, R.E. - The influence of histamine on immune and inflammatory response. Advanc. Immunol., 35: 209-268, 1984.

[3] 3. BIAGIONI, L.M.V.; ORSI, S.; CHAMMA, L.G.; SADATSUNE, T. & FRANCO, M. - Imunoglobulinas e C₃ no granuloma paracoccidióidico. Rev. Inst. Med. trop. S. Paulo, 29: 97-103, 1987.

[4] 4. BIAGIONI, L.; SOUZA, M.J.; CHAMMA, L.G.; MENDES, R.P.; MARQUES, S.A.; MOTA, N.G.S. & FRANCO, M. - 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.

[5] 5. BJERMER, L.; ENGSTRÖM-LAURENT, A.; THUNELL, M. & HÄLLGREN, R. - The mast cell and signs of pulmonary fibroblast activation in sarcoidosis. Int. Arch. Allergy, 82: 298-301, 1987.

[6] 6. BRITO, T. & FAVA NETTO, C. - Disseminated experimental South American blastomycosis of the Guinea pig; a pathologic and immunologic study. Path. et Microbiol. (Basel), 26: 29-43, 1963.

[7] 7. BURGER, D.R. & VETTO, R.M. - Hypothesis. Vascular endothelium as a major participant in T-lymphocyte immunity. Cell. Immunol., 70: 357-361, 1982.

[8] 8. CALICH, V.L.G.; KIPNIS,T.L.; MARIANO, M.; FAVA-NETO, C. & DIAS DA SILVA, W. - The activation of the complement system by Paracoccidioides brasiliensis in vitro: its opsonic effect and possible signiticance for an in vivo model of infection. Clin. Immunol. Immunopath., 12: 20-30, 1979.

[9] 9. CALICH, V.L.G.; VAZ, C.A.C. & BURGER, E. - PMN chemotactic factor produced by glass-adherent cells in the acute inflammation caused by Paracoccidioides brasiliensis Brit. J. exp. Path., 66: 57-65, 1985.

[10] 10. CARSON, F.L.; MARTIN, J.H. & LYNN, J.A.- Formalin fixation for electron microscopy. A reevaluation. Amer. J. clin. Path., 59: 365-373,1973.

[11] 11. CARVALHAES, M.S.; SILVA, W.D.; BIRMAN, E.G.; SANT'ANNA, O.A.; ABRAHAMSOHN, P. & LIBERMAN KIPNIS, T. - Experimental paracoccidioidomycosis in high and low antibody-producer mice. 1. Evolution of the disease, us correlation with the humoral immune response and the patterns of tissue lesions. Ann. Immunol. (Paris), Serie C, 137C: 127-141, 1986.

[12] 12. CLAMAN, H.N. - Mast cell, T cells and abnormal fibrosis, Immunol. Today, 6: 192-196, 1985.

[13] 13. DEXTER, T.M.; STODDART, R.W. & QUAZZAZ, S.T.A. - What are mast cells for? Nature, 291: 110-111, 1981.

[14] 14. DUNN, M.A. - Liver fibrosis in schistosomiasis. In: SLUTZKY, G.M., ed. - The Biochemistry of parasites. New York, Pergamon Press, 1980. p. 191-199.

[15] 15. FREUNDLICH, B.; BOMALASKI, J.S.; NEILSON, E. & JIMENEZ, S.A. - Regulation of fibroblast proliferation and collagen synthesis by cytokines. Immunol. Today, 7: 303-307, 1986.

[16] 16. GOIHMAN-YAHR, M.; ESSENFELD-YAHR. E.; ALBORNOZ, M.C.; YARZÁBAL, L.; GÓMEZ, M.H.; SAN MARTIN, B.; OCANTO, A.; GIL, F. & CONVIT, J. - Defect of in vitro digestive ability of polymorphonuclear leukocytes in paracoccidioidomycosis. Infect. Immun., 28: 557-566, 1980.

[17] 17. IABUKI, K. & MONTENEGRO, MR. - Experimental paracoccidioidomycosis in the Syrian hamster: Morphology, ultrastructure and correlation of lesions with presence of specific antigens and serum levels of antibodies. Mycopathologia (Deo Haag), 67: 131-141, 1979.

[18] 18. JONES, P.A. & WERB, Z. - Degradation of connective tissue matrices by macrophages. II influence of matrix composition on proteolysis of glycoproteins, elastin, and collagen by macrophages in culture. J. exp. Med., 152: 1527-1536, 1980.

[19] 19. JUNQUEIRA, L.C.U.; BIGNOLAS, G. & BRENTANI, R.R. - Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem. J., 11: 447-455, 1979.

[20] 20. JUNQUEIRA, L.C.U. & MONTES, G.S. - Biology of collagen-proteoglycan interaction. Arch, histol. jap., 46: 589-629, 1983.

[21] 21. KERR. I.B.; OLIVEIRA, P.C. & SCHAEFFER, G.V. - Influence of inoculum size on the development of intraperitoneal paracoccidioidomycosis in rats. J. infect. Dis., 149: 821, 1984.

[22] 22. KERR, I.B. SCHAEFFER, G.V. & MIRANDA, D.S. - Sex hormones and susceptibility of the rat to paracoccidioidomycosis. Mycopathologia (Den Haag), 88: 149-154, 1984.

[23] 23. KUETTNER, K.E. & KIMURA, J.H. - Proteoglycans an overview. J. cell. Biochem., 27: 327-336, 1985.

[24] 24. LENNERT, K. - Malignant lymphomas other than Hodgkin's disease. Berlin, Spring-Verlag, 1978. p.

[25] 25. LINARES, L.I. & FRIEDMAN, L. - Experimental paracoccidioidomycosis in mice. Infect. Immun., 5: 681-687,1972.

[26] 26. MACKINNON, J.E. - Pathogenesis of South American Blastomycosis. Trans. roy. Soc. trop. Med. Hyg., 53: 487-494, 1959.

[27] 27. McEWEN, J.G.; BRUMMER, E.; STEVENS, D.A. & RESTREPO, A. - Effect of murine polymorphonuclear leukocytes on the yeast form of Paracoccidioides brasiliensis Amer. J. trop. Med. Hyg., 36: 603-608, 1987.

[28] 28. McEWEN, J.G.; SUGAR, A.M.; BRUMMER, E., RESTREPO, A. & STEVENS, D.A. - Toxic effect of products of oxidative metabolism on the yeast form of Paracoccidioides brasiliensis J. med. Microbiol., 18: 423-428, 1984.

[29] 29. MOSCARDI, M. & FRANCO, M.F. - Paracoccidioidomicose experimental do camundongo. II- Infecção intraperitoneal após sensibilização prévia. Rev. Inst. Med. trop. S. Paulo, 23: 204-211, 1981.

[30] 30. 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.

[31] 31. POSTLETHWAITE, A.E.; JACKSON, B.K.; BEACHEY, E.H. & KANG, A.H. - Formation of multinucleated giant cells from human monocyte percursors. J. exp. Med., 155: 168-178, 1982.

[32] 32. RESTREPO, A. & VÉLEZ, H. - Efectos de la fagocitosis in vitro sobre el Paracoccidioides brasiliensis Sabouraudia, 13(part 1): 10-21, 1975.

[33] 33. SCHWARTZ, L.B. & AUSTEN, K.F. - Structure and function of the chemical mediators of mast cells. Progr. Allergy, 34: 271-321, 1984.

[34] 34. TEIXEIRA, G.A.; MACHADO FILHO, J. & MIRANDA, J.L. - Blastomicose sul-americana experimental. Estudo experimental em ratos com considerate relativas à patogenia das lesões. Hospital (Rio de J.), 68: 1081-1096, 1965.

[35] 35. TURK, J.L. & NARAYAMA, R.B. - The origin, morphology, and function of epithelioid cells. Immunobiology, 161:274-282, 1982.

[36] 36. WAHL, S.M. & WAHL, L.M. - Modulation of fibroblast growth and function by monokines and lymphokines. Lymphokine Res., 2: 179-201, 1981.

[37] 37. WEINBERG, J.B.; HOBBS, M.M. & MISUKONIS, M.A. - Recombinant human-interferon induces human monocyte polykaryon formation. Proc. nat. Acad. Sci. (Wash.), 81: 4554-4557, 1984.

[38] 38. WHITAKER, D. & PAPADIM1TRIOU, J. - Mesothelial healing: morphological and kinetic investigations. J. Path., 145:159-175, 1985.

[39] 39. WOLMAN, M. & KASTEN, F.H. - Polarized light microscopy in the study of the molecular structure of collagen and reticulin. Histochemistry, 85:41-49, 1986.