I~ter~fio~~ &w&for Parusit&g~ Vol. 22, No. I. pp. 1E--129. 1992 Primed in Great &it& 0
002%7519/92 $5.00 + 0.00 Pergrn* Press pit 1992 Aurtraiim Society for Pmasitology
RESEARCH NOTE
ISCHEMIA IN EXPERIMENTAL ACUTE AMEBIC LIVER ABSCESS IN HAMSTERS RUY PEREZ-TAMAYO, IRMGARD MONTFORT, EUSEBIO TELLO and ALFONSO OLIVOS Department of Experimental Medicine, Facultad de Medicina, Universidad National Autbnoma de Mexico, Mexico City, 04510 Mexico (Received 15 July 1991; accepted 7 October 1991) AbStraC&--PEREZ-TAMAYOR.,MONTFORT I.,TELLOE.and OLIVOS A.
1!#2.Isehemiainexperimental acute amebic liver abscess in hamsters. International Journalfor Parasitology 22: 125-129. In experimental acute amebic liver abscess, produced in hamsters by the intraportal inoculation of 1 x IO6axenic trophozoites of Enramoeba histolytica strain HM-1, we examined the blood perfusion of the lesions 5, lo,24 and 72 h after injection of the parasites. India ink introduced into the portal circulation filled all liver vessels but was systematically excluded from even the earher amebic lesions. The absence of serum proteinase inhibitors from the lesions may allow the participation of amebic proteinases in the causation of tissue necrosis. INDEX KEY WORDS: E&amoeba histolytica; acute liver abscess; &hernia; India ink; amebic proteinases; serum proteinase inhibitors; hamster.
THE
pathogenicity of Entamoeba histoiytica has been attributed to several factors (Orozco, Hernandez de la Cruz & Rodriguez, 1988) such as phagocytosis (Orozco, Guameros, Martinez-Palomo & Sanchez, 1983; Orozco, Rodriguez & Hernandez de la Cruz, 1988), resistance to complement (Reed, Sargeaunt 61; Braude, 1983; Mogyoros, Calef & Gitler, I986), lectinlike adhesions (Petri, Smith, Schlesinger, Murphy & Ravdin, 1987), amebopore (Lynch, Rosenberg & Gitler, 1982; Young &Cohn, 1985), proteases such as collagenase (Muiioz, Rojkind, Calderon, Tanimoto, Arias-Negrete & Martinez-Palomo, 1984; Rojkind, Rosales-Enema & Muiioz, 1988) or cysteine proteinases (Reed, Keen t McKerrow, 1989; Keene, Hidalgo, Orozco & McKerrow, 1990), and others (Lushbaugh, 1988). There is evidence that at least in the early stages of experimental amebic intestinal and liver lesions in hamsters, lysosomal enzymes released. by leukocytes and mononuclear cells destroyed by amebae may also contribute to tissue damage (Tsutsumi, Mena-Lopez, Anaya-Velazquez & Martinez-Palomo, 1984; Tsutsumi & Martinez-Palomo, 1988; MartinezPalomo, Tsutsumi, Anaya-Velazquez & GonzalezRobles, 1989). In recent papers our group has presented evidence in favor of a direct role of amebic cysteine proteinases in experimental amebic tissue necrosis (Becker, Perez-Tamayo, Montfort, Alvizouri & Perez-Montfort, 1988; Perez-Tamayo, Becker, Montfort, Ostoa-Saloma & Perez-Montfort, 1991). An unresolved problem, however, are the potent
proteinase inhibitors present in normal serum (Travis & Salvesen, 1983), especially a-Zmacroglobulin (Sottrup-Jensen, 1989) which make it at least theoretically unlikely that amebic proteinases could participate directly in tissue destruction. Some years ago (Bos, 1979), it was suggested that in areas of intimate contact between amebae and target cells or tissues, serum proteinase inhibitors would be excluded, leaving amebic proteinases free to act on the surrounding structures. This suggestion has yet to be examined. In this paper we show that ischemia is present during the early stages of experimental amebic liver abscess in hamsters, creating conditions in which amebic proteinases would not be impeded in playing a role in tissue damage. Trophozoites of Entamoeba histolytica strain HM-1 were axenically cultured in TYI-S-33 medium (Diamond, Harlow & Cunnick, 1978) at 37°C and without CO, for 72 h prior to harvesting. In order to maintain high virulence amebae were exposed weekly for 2 h to 100% fresh hamster serum (Mogyoros et al., 1986), washed and cultured again in TYI-S-33 medium. Liver abscesses were produced in anesthetized hamsters by the intraportal injection of 1 x lo6 axenic trophozoites. Four groups of four animals were prepared in order to study the perfusion of liver lesions 5,10, 24 and 72 h after the injection of amebae. At the appointed period the hamsters were anesthetized and 0.1 ml of India ink diluted in 0.9 ml of saline was slowly injected into the portal vein. The liver turned black
125
126
R. PEREZ-TAMAYOet al.
FIG. 1, Gross aspect of the hamster liver 72 h after injection of 1 x lo6 amebae and l-2 min after intraportal injection of India ink. In both the external surface (a) and the cut surface (b) the abscesses remain unstained by the India ink.
Research Note
127
FIG. 2. Microscopic thick unstained sections of the liver. a is a low power photomicrog~ph of a liver 72 h after injection of 1 x IO6amebae. The blood vessels are filled with India ink whereas the lesions remain unstained. Scale bar = 1 mm. b is a higher power photomicrograph of a liver 5 h after the injection of 1 x 10”amebae, to illustrate a very early and small lesion already devoid of blood perfusion. Scale bar = 0.2 mm.
128
R. PEREZ-TAMAYO et al.
before the injection was completed (l-2 min), and it was removed immediately. Thin slices were fixed in neutral 10% formaldehyde, dehydrated, embedded in paraffin and cut at 20 and 40 pm. The sections were mounted on microscope slides, deparaffinized, dehydrated in alcohol, cleared in xylene and covered with resin. Other sections were cut at 4 pm and stained with hematoxylin and eosin. Liver lesions were not grossly apparent 5 and 10 h after the injection of amebae and the organ appeared uniformly black. After 24 h, small (1 mm or less) white points appeared irregularly distributed on the capsular and cut surfaces of the liver. The lesions were larger (23 mm) and quite apparent after 72 h (Fig. 1). Thick unstained sections of the liver showed all blood vessels filled with India ink, the sinusoids forming a network around the cleared liver cell sheets. All amebic lesions remained free of India ink at the four times examined. This observation even applied to the smallest lesions detected by this technique, at 5 and 10 h, which were not grossly apparent (Fig. 2). Thin sections stained with H and E showed the expected microscopic development of the early stages of experimental amebic liver abscess containing apparently viable amebae. It is noteworthy that after 24 and 72 h, necrosis was the major component of the lesions and healthy trophozoites were present in the midst of tissue destruction (Tsutsumi et al., 1984; Tsutsumi & Martinez-Palomo, 1988; Montfort, Perez-Tamayo, Gonzalez Canto, Garcia de Leon, Olivos & Tello, in press). The demonstration that even early (microscopic) experimental amebic liver lesions in hamsters are excluded from the blood circulation may be relevant to their pathogenesis on at least two counts: first, the absence of serum proteinase inhibitors from the core of the lesions would leave amebic proteinases free to degrade all relevant surrounding structures; and second, ischemia in itself would represent an additional mechanism of tissue damage. It could be that the India ink technique used to demonstrate blood perfusion reflects pigment phagocytosis by Kupffer cells rather than free blood circulation. However, the interval of l-2 min between completion of the India ink injection and removal of the liver is too brief for phagocytosis to account for the massive filling of all liver blood vessels with the black marker. In addition, it may be argued that ischemia is not the same as the absence of serum proteinase inhibitors from the core of early amebic lesions; serum proteinase inhibitors could still be present in tissues devoid of blood circulation, through their interstitial diffusion, local synthesis, or both. This is an unlikely but legitimate observation, and it will have to be tested by the direct demonstration of the presence or absence of serum proteinase inhibitors in
the core of both early and late ischemic amebic lesions. work was supported in part by Grants P219CCOL891892of CONACyT and IN200289 of DGAPA. We thank Miss Aida Garcia for her expert typing Acknowledgements-This
of the manuscript. REFERENCES BECKERI., PBREZ-TAMAYO R., MONTFORTI., ALVIZOURIA. M. & PBREZ-MONTFORT R. 1988. Entamoeba histolytica: role of amebic proteinases and polymorphonuclear leukocytes in acute experimental amebiasis in the rat. Experimental Parasitology 67: 268-280. Bos H. J. 1979. Cell biological analysis of parasitehost cell interactions. II. Cell surface, phagocytosis and virulence of Entamoeba histolytica. A review. Acta Leidensia 47: 23-35. DIAMONDL. S., HARLOWD. R. & CUNNICKC. C. 1978. A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba Transactions of the Royal Society for Tropical Medicine and Hygiene 72: 431-432. KEENEW. E., HIDALG~M. E., OROZCOE. & MCKERROWJ. H. 1990. Entamoeba histolytica: correlation of the cytopathic effect of virulent trophozoites with secretion of a cysteine proteinase. Experimental Parasitology 71: 199-206. LUSHBAUGHW. B. 1988. Proteinases of Entamoeba histolytica. In: Amebiasis. Human Infection by Entamoeba histolytica (Edited by RAVDIN J. I.), pp. 219-231. John Wiley & Sons, New York. LYNCHE. C., ROSENBERG I. M. & GITLER C. 1982. An ionchannel forming protein produced by Entamoeba histolytica. European Molecular Biology Organization Journal 1: 801-804. MARTINEZ-PALOMO A., TSUTSUMIV., ANAYA-VELAZQUEZ F. & GONZALEZ-ROBLES A. 1989. Ultrastructure of experimental intestinal invasive amebiasis. American Journal of Tropical Medicine and Hygiene 41: 273-279. MOGYOROSM., CALEF E. & GITLER C. 1986. Virulence of Entamoeba histolytica correlates with the capacity to develop complement resistance. Israel Journal of Medical Sciences 22: 9 15-9 17. MONTFORT I., PBREZ-TAMAYOR., GONZALEZ CANTO A., GARCiA DE LEON M. C., OLIVOSA. & TELLOE. (in press) Entamoeba histolytica: role of amebic cysteine proteinases on the cytopathogenicity of axenic trophozoites on hamster hepatocytes in vitro. Parasitology Research. MUNOZ M. L., ROJKINDM., CALDER~N J., TANIMOTOM., ARIAS-NEGRETE S. & MARTINEZ-PALOMOA. 1984. Entamoeba histolytica: collagenolytic activity and virulence. Journal of Protozoology 31: 468470. OROZCOE., GUARNEROS G., MARTINEZ-PALOMO A. &SANCHEZ T. 1983. Entamoeba histolytica. Phagocytosis as a virulence factor. Journal of Experimental Medicine 158: 151 l-1521. OROZCOE., HERNANDEZDE LA CRUZ F. & RODRIGUEZM. A. 1988. Virulence-related properties in Entamoeba histolytica. In: Amebiasis. Human Infection by Entamoeba histolytica (Edited by RAVDIN J. I. ), pp. 314-325. John Wiley & Sons, New York. OROZCOE., RODRIGUEZM. M. & HERN~NDEZDELA CRUZ F. 1988. The role of phagocytosis in the pathogenic mech-
Research Note
129
collagenase of Entamoeba histolytica. In: Amebiasis. anism of Entamoeba histolytica. In: Amebiasis. Human Human Infection by Entamoeba histolytica (Edited by Znfecrion by Entamoeba histolytica (Edited by RAVDINJ. RAVDINJ. I.), pp. 263-272. John Wiley&Sons, New York. I.), pp. 326-338. John Wiley & Sons, New York. L. 1989. h2-Macroglobulins: structure, PEREZ-TAMAYO R., BECKERI., MONTFORT I., OSTOA-SALOMA SOITRUP-JENSEN shape, and mechanism of proteinase complex formation. P. & PBRFZ-MONTFORT R. 1991. Role of leukocytes and Journal of Biological Chemistry 264: 11,539-l 1,542. amebic proteinases in experimental rat testicular necrosis produced by Eniamoeba histolytica. Parasitology Research TRAVISJ. & SALVESEN G. S. 1983. Human plasma proteinase inhibitors. Annual Review of Biochemistry 52: 655-709. 77: 192-196. TSUTSUMIV., MENA-LOPEZR., ANAYA-VELAZQUEZ F. & PETRIW. A., JR, SMITHR. D., SCHLESINGER P. H., MURPHYC. MARTINEZ-PALOMO A. 1984. Cellular bases of experimental F. & RAVDINJ. I. 1987. Isolation of the galactose-binding amebic liver abscess formation. American Journal of lectin that mediates the in vitro adherence of Entamoeba histolytica. Journal of Clinical Investigation 80: 1238-1244. Pathology 117: 81-91. TSUTSUMIV. & MARTINEZ-PALOMO A. 1988. Inflammatory REEDS. L., SARGEAUNT P. G. & BRAUDE A. I. 1983. Resistance to lysis by human serum of pathogenic Entamoeba reaction in experimental hepatic amebiasis. An ultrahistolytica. Transactions of the Royal Society of Tropical structural study. American Journal of Pathology 130: 112119. Medicine and Hygiene 77: 248-254. REEDS. L., KEENE& MCKERROW J. H. 1989. Thiol proteinase YOUNGJ. D. -E. &COHNZ. A. 1985. Molecular mechanism of expression and pathogenicity of Entamoeba histolytica. cytotoxicity mediated by Entamoeba histolytica: characJournal of Clinical Microbiology 27: 2772-2711. terization of a pore-forming protein. Journal of Cellular ROJKIND M., ROSALES-ENCINA J. L. & MuRoz M. L. 1988. The Biochemistry 29 : 299-308.
002%7519/92 $5.00 + 0.00 Pergrn* Press pit 1992 Aurtraiim Society for Pmasitology
RESEARCH NOTE
ISCHEMIA IN EXPERIMENTAL ACUTE AMEBIC LIVER ABSCESS IN HAMSTERS RUY PEREZ-TAMAYO, IRMGARD MONTFORT, EUSEBIO TELLO and ALFONSO OLIVOS Department of Experimental Medicine, Facultad de Medicina, Universidad National Autbnoma de Mexico, Mexico City, 04510 Mexico (Received 15 July 1991; accepted 7 October 1991) AbStraC&--PEREZ-TAMAYOR.,MONTFORT I.,TELLOE.and OLIVOS A.
1!#2.Isehemiainexperimental acute amebic liver abscess in hamsters. International Journalfor Parasitology 22: 125-129. In experimental acute amebic liver abscess, produced in hamsters by the intraportal inoculation of 1 x IO6axenic trophozoites of Enramoeba histolytica strain HM-1, we examined the blood perfusion of the lesions 5, lo,24 and 72 h after injection of the parasites. India ink introduced into the portal circulation filled all liver vessels but was systematically excluded from even the earher amebic lesions. The absence of serum proteinase inhibitors from the lesions may allow the participation of amebic proteinases in the causation of tissue necrosis. INDEX KEY WORDS: E&amoeba histolytica; acute liver abscess; &hernia; India ink; amebic proteinases; serum proteinase inhibitors; hamster.
THE
pathogenicity of Entamoeba histoiytica has been attributed to several factors (Orozco, Hernandez de la Cruz & Rodriguez, 1988) such as phagocytosis (Orozco, Guameros, Martinez-Palomo & Sanchez, 1983; Orozco, Rodriguez & Hernandez de la Cruz, 1988), resistance to complement (Reed, Sargeaunt 61; Braude, 1983; Mogyoros, Calef & Gitler, I986), lectinlike adhesions (Petri, Smith, Schlesinger, Murphy & Ravdin, 1987), amebopore (Lynch, Rosenberg & Gitler, 1982; Young &Cohn, 1985), proteases such as collagenase (Muiioz, Rojkind, Calderon, Tanimoto, Arias-Negrete & Martinez-Palomo, 1984; Rojkind, Rosales-Enema & Muiioz, 1988) or cysteine proteinases (Reed, Keen t McKerrow, 1989; Keene, Hidalgo, Orozco & McKerrow, 1990), and others (Lushbaugh, 1988). There is evidence that at least in the early stages of experimental amebic intestinal and liver lesions in hamsters, lysosomal enzymes released. by leukocytes and mononuclear cells destroyed by amebae may also contribute to tissue damage (Tsutsumi, Mena-Lopez, Anaya-Velazquez & Martinez-Palomo, 1984; Tsutsumi & Martinez-Palomo, 1988; MartinezPalomo, Tsutsumi, Anaya-Velazquez & GonzalezRobles, 1989). In recent papers our group has presented evidence in favor of a direct role of amebic cysteine proteinases in experimental amebic tissue necrosis (Becker, Perez-Tamayo, Montfort, Alvizouri & Perez-Montfort, 1988; Perez-Tamayo, Becker, Montfort, Ostoa-Saloma & Perez-Montfort, 1991). An unresolved problem, however, are the potent
proteinase inhibitors present in normal serum (Travis & Salvesen, 1983), especially a-Zmacroglobulin (Sottrup-Jensen, 1989) which make it at least theoretically unlikely that amebic proteinases could participate directly in tissue destruction. Some years ago (Bos, 1979), it was suggested that in areas of intimate contact between amebae and target cells or tissues, serum proteinase inhibitors would be excluded, leaving amebic proteinases free to act on the surrounding structures. This suggestion has yet to be examined. In this paper we show that ischemia is present during the early stages of experimental amebic liver abscess in hamsters, creating conditions in which amebic proteinases would not be impeded in playing a role in tissue damage. Trophozoites of Entamoeba histolytica strain HM-1 were axenically cultured in TYI-S-33 medium (Diamond, Harlow & Cunnick, 1978) at 37°C and without CO, for 72 h prior to harvesting. In order to maintain high virulence amebae were exposed weekly for 2 h to 100% fresh hamster serum (Mogyoros et al., 1986), washed and cultured again in TYI-S-33 medium. Liver abscesses were produced in anesthetized hamsters by the intraportal injection of 1 x lo6 axenic trophozoites. Four groups of four animals were prepared in order to study the perfusion of liver lesions 5,10, 24 and 72 h after the injection of amebae. At the appointed period the hamsters were anesthetized and 0.1 ml of India ink diluted in 0.9 ml of saline was slowly injected into the portal vein. The liver turned black
125
126
R. PEREZ-TAMAYOet al.
FIG. 1, Gross aspect of the hamster liver 72 h after injection of 1 x lo6 amebae and l-2 min after intraportal injection of India ink. In both the external surface (a) and the cut surface (b) the abscesses remain unstained by the India ink.
Research Note
127
FIG. 2. Microscopic thick unstained sections of the liver. a is a low power photomicrog~ph of a liver 72 h after injection of 1 x IO6amebae. The blood vessels are filled with India ink whereas the lesions remain unstained. Scale bar = 1 mm. b is a higher power photomicrograph of a liver 5 h after the injection of 1 x 10”amebae, to illustrate a very early and small lesion already devoid of blood perfusion. Scale bar = 0.2 mm.
128
R. PEREZ-TAMAYO et al.
before the injection was completed (l-2 min), and it was removed immediately. Thin slices were fixed in neutral 10% formaldehyde, dehydrated, embedded in paraffin and cut at 20 and 40 pm. The sections were mounted on microscope slides, deparaffinized, dehydrated in alcohol, cleared in xylene and covered with resin. Other sections were cut at 4 pm and stained with hematoxylin and eosin. Liver lesions were not grossly apparent 5 and 10 h after the injection of amebae and the organ appeared uniformly black. After 24 h, small (1 mm or less) white points appeared irregularly distributed on the capsular and cut surfaces of the liver. The lesions were larger (23 mm) and quite apparent after 72 h (Fig. 1). Thick unstained sections of the liver showed all blood vessels filled with India ink, the sinusoids forming a network around the cleared liver cell sheets. All amebic lesions remained free of India ink at the four times examined. This observation even applied to the smallest lesions detected by this technique, at 5 and 10 h, which were not grossly apparent (Fig. 2). Thin sections stained with H and E showed the expected microscopic development of the early stages of experimental amebic liver abscess containing apparently viable amebae. It is noteworthy that after 24 and 72 h, necrosis was the major component of the lesions and healthy trophozoites were present in the midst of tissue destruction (Tsutsumi et al., 1984; Tsutsumi & Martinez-Palomo, 1988; Montfort, Perez-Tamayo, Gonzalez Canto, Garcia de Leon, Olivos & Tello, in press). The demonstration that even early (microscopic) experimental amebic liver lesions in hamsters are excluded from the blood circulation may be relevant to their pathogenesis on at least two counts: first, the absence of serum proteinase inhibitors from the core of the lesions would leave amebic proteinases free to degrade all relevant surrounding structures; and second, ischemia in itself would represent an additional mechanism of tissue damage. It could be that the India ink technique used to demonstrate blood perfusion reflects pigment phagocytosis by Kupffer cells rather than free blood circulation. However, the interval of l-2 min between completion of the India ink injection and removal of the liver is too brief for phagocytosis to account for the massive filling of all liver blood vessels with the black marker. In addition, it may be argued that ischemia is not the same as the absence of serum proteinase inhibitors from the core of early amebic lesions; serum proteinase inhibitors could still be present in tissues devoid of blood circulation, through their interstitial diffusion, local synthesis, or both. This is an unlikely but legitimate observation, and it will have to be tested by the direct demonstration of the presence or absence of serum proteinase inhibitors in
the core of both early and late ischemic amebic lesions. work was supported in part by Grants P219CCOL891892of CONACyT and IN200289 of DGAPA. We thank Miss Aida Garcia for her expert typing Acknowledgements-This
of the manuscript. REFERENCES BECKERI., PBREZ-TAMAYO R., MONTFORTI., ALVIZOURIA. M. & PBREZ-MONTFORT R. 1988. Entamoeba histolytica: role of amebic proteinases and polymorphonuclear leukocytes in acute experimental amebiasis in the rat. Experimental Parasitology 67: 268-280. Bos H. J. 1979. Cell biological analysis of parasitehost cell interactions. II. Cell surface, phagocytosis and virulence of Entamoeba histolytica. A review. Acta Leidensia 47: 23-35. DIAMONDL. S., HARLOWD. R. & CUNNICKC. C. 1978. A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba Transactions of the Royal Society for Tropical Medicine and Hygiene 72: 431-432. KEENEW. E., HIDALG~M. E., OROZCOE. & MCKERROWJ. H. 1990. Entamoeba histolytica: correlation of the cytopathic effect of virulent trophozoites with secretion of a cysteine proteinase. Experimental Parasitology 71: 199-206. LUSHBAUGHW. B. 1988. Proteinases of Entamoeba histolytica. In: Amebiasis. Human Infection by Entamoeba histolytica (Edited by RAVDIN J. I.), pp. 219-231. John Wiley & Sons, New York. LYNCHE. C., ROSENBERG I. M. & GITLER C. 1982. An ionchannel forming protein produced by Entamoeba histolytica. European Molecular Biology Organization Journal 1: 801-804. MARTINEZ-PALOMO A., TSUTSUMIV., ANAYA-VELAZQUEZ F. & GONZALEZ-ROBLES A. 1989. Ultrastructure of experimental intestinal invasive amebiasis. American Journal of Tropical Medicine and Hygiene 41: 273-279. MOGYOROSM., CALEF E. & GITLER C. 1986. Virulence of Entamoeba histolytica correlates with the capacity to develop complement resistance. Israel Journal of Medical Sciences 22: 9 15-9 17. MONTFORT I., PBREZ-TAMAYOR., GONZALEZ CANTO A., GARCiA DE LEON M. C., OLIVOSA. & TELLOE. (in press) Entamoeba histolytica: role of amebic cysteine proteinases on the cytopathogenicity of axenic trophozoites on hamster hepatocytes in vitro. Parasitology Research. MUNOZ M. L., ROJKINDM., CALDER~N J., TANIMOTOM., ARIAS-NEGRETE S. & MARTINEZ-PALOMOA. 1984. Entamoeba histolytica: collagenolytic activity and virulence. Journal of Protozoology 31: 468470. OROZCOE., GUARNEROS G., MARTINEZ-PALOMO A. &SANCHEZ T. 1983. Entamoeba histolytica. Phagocytosis as a virulence factor. Journal of Experimental Medicine 158: 151 l-1521. OROZCOE., HERNANDEZDE LA CRUZ F. & RODRIGUEZM. A. 1988. Virulence-related properties in Entamoeba histolytica. In: Amebiasis. Human Infection by Entamoeba histolytica (Edited by RAVDIN J. I. ), pp. 314-325. John Wiley & Sons, New York. OROZCOE., RODRIGUEZM. M. & HERN~NDEZDELA CRUZ F. 1988. The role of phagocytosis in the pathogenic mech-
Research Note
129
collagenase of Entamoeba histolytica. In: Amebiasis. anism of Entamoeba histolytica. In: Amebiasis. Human Human Infection by Entamoeba histolytica (Edited by Znfecrion by Entamoeba histolytica (Edited by RAVDINJ. RAVDINJ. I.), pp. 263-272. John Wiley&Sons, New York. I.), pp. 326-338. John Wiley & Sons, New York. L. 1989. h2-Macroglobulins: structure, PEREZ-TAMAYO R., BECKERI., MONTFORT I., OSTOA-SALOMA SOITRUP-JENSEN shape, and mechanism of proteinase complex formation. P. & PBRFZ-MONTFORT R. 1991. Role of leukocytes and Journal of Biological Chemistry 264: 11,539-l 1,542. amebic proteinases in experimental rat testicular necrosis produced by Eniamoeba histolytica. Parasitology Research TRAVISJ. & SALVESEN G. S. 1983. Human plasma proteinase inhibitors. Annual Review of Biochemistry 52: 655-709. 77: 192-196. TSUTSUMIV., MENA-LOPEZR., ANAYA-VELAZQUEZ F. & PETRIW. A., JR, SMITHR. D., SCHLESINGER P. H., MURPHYC. MARTINEZ-PALOMO A. 1984. Cellular bases of experimental F. & RAVDINJ. I. 1987. Isolation of the galactose-binding amebic liver abscess formation. American Journal of lectin that mediates the in vitro adherence of Entamoeba histolytica. Journal of Clinical Investigation 80: 1238-1244. Pathology 117: 81-91. TSUTSUMIV. & MARTINEZ-PALOMO A. 1988. Inflammatory REEDS. L., SARGEAUNT P. G. & BRAUDE A. I. 1983. Resistance to lysis by human serum of pathogenic Entamoeba reaction in experimental hepatic amebiasis. An ultrahistolytica. Transactions of the Royal Society of Tropical structural study. American Journal of Pathology 130: 112119. Medicine and Hygiene 77: 248-254. REEDS. L., KEENE& MCKERROW J. H. 1989. Thiol proteinase YOUNGJ. D. -E. &COHNZ. A. 1985. Molecular mechanism of expression and pathogenicity of Entamoeba histolytica. cytotoxicity mediated by Entamoeba histolytica: characJournal of Clinical Microbiology 27: 2772-2711. terization of a pore-forming protein. Journal of Cellular ROJKIND M., ROSALES-ENCINA J. L. & MuRoz M. L. 1988. The Biochemistry 29 : 299-308.
