Immunohistochemical localization of excretory/secretory antigens in adult Ancylostoma caninum using monoclonal antibodies and infected human sera

Abstract

Human eosinophilic enteritis (EE) may result from hypersensitivity to the excretory/secretory (ES) antigens of adult Ancylostoma caninum. The origin of several antigens were identified by probing adult A. caninum with mouse monoclonal antibodies (MoAbs), sera from mice vaccinated with ES antigens and sera from human EE patients. Six MoAbs (AC/ES 1-6) were produced against ES antigens, two being IgG3 and four IgM. Western blots demonstrated four different antigen specificities: MoAb AC/ES 1 bound strongly to an ES product at about 30 kDa; AC/ES 2 recognized a broad band ranging from 50-200 kDa; AC/ES 3, AC/ES 5 and AC/ES 6 reacted at about 68 kDa, and AC/ES 4 at about 97 kDa. Sections of formalin-fixed, paraffin embedded adult A. caninum were then incubated with these MoAbs and immunostained by the peroxidase-anti-peroxidase (PAP) technique. The target epitope of MoAb AC/ES 1 was found mainly in the oesophageal, amphidial and excretory glands; AC/ES 2 reacted weakly with many structures in the sections; AC/ES 3, AC/ES 5 and AC/ES 6 were specific for excretory glands only, and AC/ES 4 bound to amphidial glands. Sera from immunized mice reacted with all three (especially the excretory) glands and the cuticle. In an indirect assay, worm sections probes with three human EE patient sera demonstrated maximal staining in the amphidial glands. Our findings confirm that ES products of A. caninum include immunogenic glandular secretions which may be involved in the pathogenesis of human EE.

Cleavage of hemoglobin by hookworm cathepsin D aspartic proteases and its potential contribution to host specificity

“…Intact, live adult A. caninum and N. americanus worms obtained from the small intestine of freshly killed hosts were washed in PBS and fixed by placement in phosphate‐buffered formalin (70% formalin, 0.4% Na2H2PO4, pH 7.4), after which they were heated in a microwave on high power for 40 s. Worms were then washed twice for 5 min in PBS, pH 7.2, dehydrated through a graded series of ethanol (70–100%), cleared in methyl salicylate, and embedded in paraffin via standard preparative procedures (22). Fixed hookworm sections of 5 μm were deparaffinized, rehydrated in a graded series of alcohols, and reacted with antisera as previously described (23). Sections were then washed, counterstained with hematoxylin for 5 min and Scott's Blue, dehydrated in an ethanol series (70–100%), cleared in 100% toluene, and mounted in DPX for examination by light microscopy.…”

Hookworm Aspartic Protease,Na‐APR‐2, Cleaves Human Hemoglobin and Serum Proteins in a Host‐Specific Fashion

“…Five micrometer sections were deparaffinized, rehydrated in graded alcohols, and reacted with mouse antiserum to Na-APR-2, followed by peroxidase-conjugated anti-mouse IgG. Antibody binding was visualized with 3,3 -diaminobenzidine (DAB) substrate, as described elsewhere [18]. Sections then were washed, counterstained with hemotoxylin and Scott's Blue for 5 min, dehydrated in an ethanol series (70%-100%), cleared in 100% toluene, and mounted in DPX for examination by light microscopy.…”

Intestinal obstruction by eosinophilic jejunitis

“…Aunque su etiología no está resuelta, se ha intentado clásicamente demostrar su relación con la alergia alimentaria sin conseguirlo. Sí es más evidente su asociación a infestación por Ancylostoma caninum, descrito sobre todo en series clínicas australianas, donde esta enfermedad es más prevalente que en otras zonas del planeta y se ha estudiado bastante esta patología, sobre todo por Croese y Prociv (3)(4)(5)(6). Excepcionalmente se ha descrito también asociado al tratamiento con carbamacepina (7).…”

“…Its association with an infestation by Ancylostoma caninum is more evident. This disease has been widely studied, particularly by Croese and Prociv (3)(4)(5)(6) in Australia where this disease is specially frequent. This entity has also been described in patients treated with carbamazepine (7).…”