Archives of Medical Research 31 (2000) S242–S244 0188-4409/00 $–see front matter. Copyright © 2000 IMSS. Published by Elsevier Science Inc. PII S0188-4409(00)00182-X Entamoeba histolytica: Localization of the Gal/GalNAc Adherence Lectin in Experimental Amebic Liver Abscess Judith Pacheco-Yépez,* Rafael Campos-Rodríguez,*** Jesús Serrano-Luna,** Martha Espinosa-Cantellano,* William A. Petri, Jr,**** Víctor Tsutsumi* and Mineko Shibayama* *Departamento de Patología Experimental **Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del I.P.N. (Cinvestav), Mexico City, Mexico ***Departamento de Bioquímica, Escuela Superior de Medicina, Instituto Politécnico Nacional (IPN), Mexico City, Mexico ****Department of Medicine, University of Virginia, Charlottesville, VA, USA Key Words: Entamoeba histolytica, Gal/GalNAc lectin, Amebic liver abscess, Immunohistochemistry. Introduction Intestinal invasive amebiasis is characterized by ulceration of the colonic epithelium, while amebic liver abscess, the main extraintestinal form of the infection, produces large necrotic areas that can be fatal unless properly treated. De- spite great advances in the knowledge of the parasite, little is known about the pathogenic mechanisms that participate in the production of these lesions in the host. In vitro, the cytopathic effect of amebas involves adher- ence, contact-dependent lysis, and phagocytosis. Several molecules seem to play an important role in these events, in- cluding lectins, pore-forming peptides, and proteases. One of the best characterized molecules is the galactose/N-acetyl- D-galactosamine (Gal/GalNAc)-inhibitable lectin, which par- ticipates in the in vitro adhesion to human erythrocytes, neu- trophils, epithelial cells, and colonic mucins (1). In addition to its role in amebic adherence, the lectin may also partici- pate in the cytolytic event, because contact-dependent target cell lysis is reduced in the presence of galactose, and a mon- oclonal antibody against the heavy subunit is capable of in- hibiting cytolysis without blocking adherence (2). Further- more, the adhesin binds to purified C8 and C9 components of complement and blocks the assembly of the membrane attack complex on the amebic plasma membrane, suggest- ing a role in mediating resistance to complement lysis through components C5b–9 (3). Because the Gal/GalNAc lectin plays an important role in the in vitro cytopathic effect, the aim of this project was to determine the participation of this molecule in the in vivo experimental infection. We present here the localization of the Gal/GalNAc lectin in the amebic liver abscess at differ- ent times of evolution. Materials and Methods Amebas. Entamoeba histolytica trophozoites strain HM- 1:IMSS were axenically cultured at 36°C in BI-S-33 me- dium. Parasites were harvested after 72 h incubation by chilling the culture tubes to 4°C and centrifuging at 160  g for 5 min. Production of amebic liver abscess. Two-month-old male adult golden hamsters (Mesocricetus auratus) weighing ap- proximately 100 g were intraportally inoculated with 0.1 mL culture medium containing 5  10 5 E. histolytica tro- phozoites. Groups of five animals were sacrificed at 6, 12, 24, and 48 h postinoculation. Lesions associated with nor- mal hepatic tissue were fixed, embedded in paraffin, and stained with hematoxilin and eosin to identify representa- tive samples of each timepoint to be processed for immuno- histochemistry. Immunohistochemistry. Sections 6–7 m-thick were mounted on glass slides covered with silane. After dewaxing with xy- lene, endogenous peroxidase activity was blocked by incu- bation with 0.3% H 2 O 2 in methanol for 1 h, and washed with phosphate-buffered saline (PBS). Nonspecific reaction sites were blocked for 1 h at room temperature with 10% fetal bo- vine serum/3% rabbit serum in 0.05% PBS-Tween (PBS-T). Slides were incubated overnight at 4°C with a rabbit poly- clonal anti-Gal/GalNAc lectin antibody or with a rabbit anti- CD8 antibody (controls) diluted 1:100 in PBS-T. After sev- eral washings in PBS-T, samples were incubated with a per- oxidase-labeled goat antirabbit IgG antibody for 1 h at room temperature. Peroxidase activity was detected by adding di- Address reprint requests to: Mineko Shibayama, Departamento de Patología Experimental, Cinvestav, Av. Instituto Politécnico Nacional #2508, Col. San Pedro Zacatenco, 07369 México, D.F., México. Tel.: (+525) 747- 7107; FAX: (+525) 747-7107; E-mail: mineko@mail.cinvestav.mx Presenting author: Judith Pacheco-Yépez.