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Circulating antibodies to histolysain, the major cysteine proteinase of Entamoeba histolytica, in amoebic liver abscess patients L. M. OSORIO*, T. PlCOf and A. LUACESf Departamento de Parasitologia, Institute de Medicina Tropical 'Pedro Kouri' (IPK), Ciudad de la Habana, Cuba (Received 21 October 1991; revised 12 March 1992; accepted 12 March 1992)
SUMMARY
A solid-phase enzyme immunoassay (EIA) was used to detect circulating antibodies to histolysain, the major cysteine proteinase of Entamoeba histolytica. Serum samples from 40 healthy controls, 33 asymptomatic E. histolytica cyst passers and 22 patients with amoebic liver abscess were tested. Antibodies to histolysain were found in 727 ° 0 of cases of amoebic liver abscess, 18" 1 ° 0 of the cyst passers and 2'5 ° 0 of healthy controls, which suggests that a humoral immune response is induced by histolysain during amoebic liver abscess. Key words: histolysain, Entamoeba histolytica, antibodies, amoebiasis, liver abscess, immunoassay.
INTRODUCTION
Some degree of protective immunity may develop following invasive human infection by Entamoeba histolytica (De Leon, 1970; Sepulveda & Martinez Palomo, 1982), and the pharmacological cure of amoebic colitis or liver abscess may be followed by an acquired state of immunity. However, the specific immune responses involved are still not well understood. Identification of specific E. histolytica components whose function is crucial in disease pathogenesis might provide opportunities for a directed host immune response. It is generally accepted that the histolytic activity of E. histolytica is related to the action of proteolytic enzymes, which may play a considerable role in inducing tissue lesions and tissue invasion by the parasite (Lushbaugh & Pittman, 1984; Gitler & Mirelman, 1986; Becker et al. 1988). A correlation has been observed between invasive activity and the amount of cysteine proteinase present in the strains of E. histolytica exhibiting various degrees of virulence (Gadasi & Kessler, 1983; Gadasi & Kobiler, 1983; Lushbaugh et al. 1984; Lushbaugh, Hofbauer & Pittman, 1985). There are several reports of partial or complete purification of cysteine proteinases of this protozoan (McLaughlin & Faubert, 1977; Scholze & Werries, 1984, 1986; Lushbaugh et al. 1985; Keene et al. 1986; Perez-Montfort et al. 1987). We have recently * Present address : Departamento de Immunoquimica, Instituto Nacional de Oncologia y Radiobiologi'a, Calle 29 y E Vedado, C. Habana, Cuba. t Present address: CODEME S.A., Calle Cero No. 114, Miramar, C. Habana, Cuba. Parasitology (1992), 105, 207-210
Printed in Great Britain
purified and characterized the major cysteine proteinase of E. histolytica, which we initially named histolysin (Luaces & Barrett, 1988), and now term histolysain, in line with the convention of using the '-ain' ending for the names of cysteine proteinases. This protein (Mr 29000) probably plays a key role in tissue invasion, since it degrades components of extracellular matrix and also causes rounding and detachment of cells in culture. The purpose of the present study was to determine whether histolysain is immunogenic, by detecting anti-histolysain antibodies in sera from patients with invasive and non-invasive amoebiasis. MATERIALS AND METHODS
Human sera Sera of normal healthy controls (40 subjects), asymptomatic E. histolytica cyst passers (33 subjects), and patients with amoebic liver abscess (22 subjects) were used. The normal healthy control group included males and females without recent or past history of amoebic disease and with stool examinations negative for E. histolytica. Microscopically diagnosed cyst passers were all asymptomatic. The diagnosis of both controls and cyst passers was confirmed by use of immuno-enzymatic assay for histolysain in stools (ENZYMEBA) as described in the accompanying paper (Luaces, Pico & Barrett, 1992). Sera of amoebic liver abscess patients were kindly provided by Dr Esther Orozco at Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, Mexico City, Mexico. The diagnosis of amoebic liver abscess was made according to WHO recommendations (WHO, 1969).
L. M. Osorio, T. Pico and A. Luaces
We also tested serum samples from 5 patients with ascariasis, 3 with trichuriasis, 3 with filariasis, 8 with giardiasis, 6 with schistosomiasis, 5 with hookworm and 3 with fascioliasis, all diagnosed by standard parasitological methods. Blood samples were drawn, serum was separated after centrifugation, and the samples stored at — 20 °C until use. Amoebic serology was by immunofluorescence assay (IFA) as described below.
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for 3 h at 4 °C. The wells were then washed 5 times with PBS-T and incubated with 100/
Circulating antibodies to histolysain, the major cysteine proteinase of Entamoeba histolytica, in amoebic liver abscess patients L. M. OSORIO*, T. PlCOf and A. LUACESf Departamento de Parasitologia, Institute de Medicina Tropical 'Pedro Kouri' (IPK), Ciudad de la Habana, Cuba (Received 21 October 1991; revised 12 March 1992; accepted 12 March 1992)
SUMMARY
A solid-phase enzyme immunoassay (EIA) was used to detect circulating antibodies to histolysain, the major cysteine proteinase of Entamoeba histolytica. Serum samples from 40 healthy controls, 33 asymptomatic E. histolytica cyst passers and 22 patients with amoebic liver abscess were tested. Antibodies to histolysain were found in 727 ° 0 of cases of amoebic liver abscess, 18" 1 ° 0 of the cyst passers and 2'5 ° 0 of healthy controls, which suggests that a humoral immune response is induced by histolysain during amoebic liver abscess. Key words: histolysain, Entamoeba histolytica, antibodies, amoebiasis, liver abscess, immunoassay.
INTRODUCTION
Some degree of protective immunity may develop following invasive human infection by Entamoeba histolytica (De Leon, 1970; Sepulveda & Martinez Palomo, 1982), and the pharmacological cure of amoebic colitis or liver abscess may be followed by an acquired state of immunity. However, the specific immune responses involved are still not well understood. Identification of specific E. histolytica components whose function is crucial in disease pathogenesis might provide opportunities for a directed host immune response. It is generally accepted that the histolytic activity of E. histolytica is related to the action of proteolytic enzymes, which may play a considerable role in inducing tissue lesions and tissue invasion by the parasite (Lushbaugh & Pittman, 1984; Gitler & Mirelman, 1986; Becker et al. 1988). A correlation has been observed between invasive activity and the amount of cysteine proteinase present in the strains of E. histolytica exhibiting various degrees of virulence (Gadasi & Kessler, 1983; Gadasi & Kobiler, 1983; Lushbaugh et al. 1984; Lushbaugh, Hofbauer & Pittman, 1985). There are several reports of partial or complete purification of cysteine proteinases of this protozoan (McLaughlin & Faubert, 1977; Scholze & Werries, 1984, 1986; Lushbaugh et al. 1985; Keene et al. 1986; Perez-Montfort et al. 1987). We have recently * Present address : Departamento de Immunoquimica, Instituto Nacional de Oncologia y Radiobiologi'a, Calle 29 y E Vedado, C. Habana, Cuba. t Present address: CODEME S.A., Calle Cero No. 114, Miramar, C. Habana, Cuba. Parasitology (1992), 105, 207-210
Printed in Great Britain
purified and characterized the major cysteine proteinase of E. histolytica, which we initially named histolysin (Luaces & Barrett, 1988), and now term histolysain, in line with the convention of using the '-ain' ending for the names of cysteine proteinases. This protein (Mr 29000) probably plays a key role in tissue invasion, since it degrades components of extracellular matrix and also causes rounding and detachment of cells in culture. The purpose of the present study was to determine whether histolysain is immunogenic, by detecting anti-histolysain antibodies in sera from patients with invasive and non-invasive amoebiasis. MATERIALS AND METHODS
Human sera Sera of normal healthy controls (40 subjects), asymptomatic E. histolytica cyst passers (33 subjects), and patients with amoebic liver abscess (22 subjects) were used. The normal healthy control group included males and females without recent or past history of amoebic disease and with stool examinations negative for E. histolytica. Microscopically diagnosed cyst passers were all asymptomatic. The diagnosis of both controls and cyst passers was confirmed by use of immuno-enzymatic assay for histolysain in stools (ENZYMEBA) as described in the accompanying paper (Luaces, Pico & Barrett, 1992). Sera of amoebic liver abscess patients were kindly provided by Dr Esther Orozco at Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, Mexico City, Mexico. The diagnosis of amoebic liver abscess was made according to WHO recommendations (WHO, 1969).
L. M. Osorio, T. Pico and A. Luaces
We also tested serum samples from 5 patients with ascariasis, 3 with trichuriasis, 3 with filariasis, 8 with giardiasis, 6 with schistosomiasis, 5 with hookworm and 3 with fascioliasis, all diagnosed by standard parasitological methods. Blood samples were drawn, serum was separated after centrifugation, and the samples stored at — 20 °C until use. Amoebic serology was by immunofluorescence assay (IFA) as described below.
208
for 3 h at 4 °C. The wells were then washed 5 times with PBS-T and incubated with 100/
