INFECTION AND IMMUNITY, Nov. 2007, p. 5158–5166 0019-9567/07/$08.00⫹0 doi:10.1128/IAI.01175-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved.
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Taenia solium Oncosphere Adhesion to Intestinal Epithelial and Chinese Hamster Ovary Cells In Vitro䌤 Manuela Verastegui,1 Robert H. Gilman,1,2,7* Yanina Arana,1 Dylan Barber,3 Jeanette Vela´squez,1 Marilu Farfa´n,1 Nancy Chile,1 Jon C. Kosek,4 Margaret Kosek,2 Hector H. Garcia,1,5 Armando Gonzalez,6 and the Cysticercosis Working Group in Peru Department of Microbiology, Universidad Peruana Cayetano Heredia, P.O. Box 5045, Lima, Peru1; Department of International Health, Johns Hopkins University, Bloomberg School of Hygiene and Public Health, 615 N. Wolfe Street, Room W#5515, Baltimore, Maryland 212052; Faculty of Veterinary Science, University of Melbourne, 250 Princes Highway, Werribe, Victoria 3030, Australia3; Department of Pathology, Stanford University School of Medicine, Stanford, California 943054; Cysticercosis Unit, Instituto de Ciencias Neurologicas, Jr. Ancash 1271, Barrios Altos, Lima, Peru5; Public Health Section, School of Veterinary Medicine, Universidad Nacional Mayor de San Marcos, Apartado 03-5113, Lima 03, Peru6; and AB PRISMA, Calle Carlos Gonzales 251, San Miguel, Lima 32, Peru7 Received 26 July 2006/Returned for modification 14 September 2006/Accepted 20 June 2007
The specific mechanisms underlying Taenia solium oncosphere adherence and penetration in the host have not been studied previously. We developed an in vitro adhesion model assay to evaluate the mechanisms of T. solium oncosphere adherence to the host cells. The following substrates were used: porcine intestinal mucosal scrapings (PIMS), porcine small intestinal mucosal explants (PSIME), Chinese hamster ovary cells (CHO cells), epithelial cells from ileocecal colorectal adenocarcinoma (HCT-8 cells), and epithelial cells from colorectal adenocarcinoma (Caco-2 cells). CHO cells were used to compare oncosphere adherence to fixed and viable cells, to determine the optimum time of oncosphere incubation, to determine the role of sera and monolayer cell maturation, and to determine the effect of temperature on oncosphere adherence. Light microscopy, scanning microscopy, and transmission microscopy were used to observe morphological characteristics of adhered oncospheres. This study showed in vitro adherence of activated T. solium oncospheres to PIMS, PSIME, monolayer CHO cells, Caco-2 cells, and HCT-8 cells. The reproducibility of T. solium oncosphere adherence was most easily measured with CHO cells. Adherence was enhanced by serum-binding medium with >5% fetal bovine serum, which resulted in a significantly greater number of oncospheres adhering than the number adhering when serum at a concentration less than 2.5% was used (P < 0.05). Oncosphere adherence decreased with incubation of cells at 4°C compared with the adherence at 37°C. Our studies also demonstrated that T. solium oncospheres attach to cells with elongated microvillus processes and that the oncospheres expel external secretory vesicles that have the same oncosphere processes. the eggs in the host small intestine and then activated by the action of intestinal enzymes and bile salts. In order to penetrate through intestinal cells, the oncosphere must first adhere. Little is known about the process of oncosphere adhesion, which is required for penetration and establishment of the larval cestode in the host. The mechanisms by which T. solium oncospheres infect host tissues are not known. One of the initial steps of infection by many microorganisms involves adhesion to host cells. The proteins involved in the adherence mechanism can be exploited as targets for developing vaccines that might inhibit parasite adherence and consequently infection. Adhesion of pathogens to host cells is the first step in invasion of all infectious disease pathogens. For instance, Entamoeba histolytica, Trichomonas sp., and Trypanosoma cruzi attach to mammalian cells via specific adhesins (2, 6, 10, 28). Similar mechanisms of adherence have been observed for Helicobacter pylori, Actinomyces naeslundii, and Pseudomonas aeruginosa (3, 7, 37). In order to focus on the initial adhesion of T. solium oncospheres, an in vitro adhesion assay model was developed to measure T. solium oncosphere adhesion using three different substrates: (i) porcine intestinal mucosal scrapings
Cysticercosis is a common disease in areas of the third world where pigs are raised. The life cycle of Taenia solium includes the pig as the normal intermediate host that harbors the larval vesicles or cysticerci. Humans can also serve as accidental intermediate hosts through ingestion of tapeworm eggs (1, 13, 24). Human cysticercosis is a parasitic disease caused by larvae of the cestode T. solium. It is acquired when eggs released by the adult tapeworm located in the human small intestine are passed in human feces and accidentally ingested. Human cysticercosis is the most common helminthic parasitic illness affecting the central nervous system. It is especially a problem in countries where the sanitary infrastructure is deficient. In rural areas of Latin America, between 5 and 20% of the population show circulating antibodies to T. solium (4, 12, 14). T. solium eggs contain an oncosphere that is released from
* Corresponding author. Mailing address: Department of International Health, Johns Hopkins University, Bloomberg School of Hygiene and Public Health, 615 N. Wolfe Street, Room W#5515, Baltimore, MD 21205. Phone: (410) 614-3959. Fax: (410) 614-6060. E-mail:
[email protected]. 䌤 Published ahead of print on 13 August 2007. 5158
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(PIMS) (cells plus mucin), (ii) porcine small intestinal mucosal explants (PSIME), and (iii) monolayer cells, including Chinese hamster ovary (CHO) cells (CHO-K1 cells), epithelial cells from ileocecal colorectal adenocarcinoma (HCT-8 cells), and epithelial cells from colorectal adenocarcinoma (Caco-2 cells). MATERIALS AND METHODS T. solium oncosphere preparation. Adult T. solium tapeworms were collected from newly diagnosed patients as previously reported (18, 36). Species were differentiated by histology (parasite morphology) and PCR-restriction fragment length polymorphism analysis (PCR with restriction enzyme analysis) (22). The proglottids were collected by sieving, washed thoroughly with distilled water, and stored in 25% glycerol supplemented with penicillin (1,000 U/ml), gentamicin (100 g/ml), streptomycin (1 mg/ml), and amphotericin B (20 g/ml) at 4°C until they were used. The eggs were obtained from gravid proglottids by gentle homogenization (using a manual homogenizer) in water. Eggs were then washed three times in distilled water by centrifugation at 1,575 ⫻ g prior to hatching. In vitro hatching of oncospheres was performed using 0.75% sodium hypochlorite (Mallinckrodt Baker, Inc., Phillipsburg, NJ) in water for 10 min at 4°C as previously described (20, 33, 35). Oncospheres were then washed three times in RPMI 1640 medium by centrifugation at 1,575 ⫻ g and activated with artificial intestinal fluid (1 g pancreatin [Sigma Chemical Co., St. Louis, MO], 200 mg Na2CO3, and 1 ml pig bile, with the volume adjusted to 100 ml with RPMI 1640 medium [pH 8.04]) by incubation at 37°C for 1 h (23). After activation, oncospheres were washed three times in RPMI 1640 medium, resuspended in RPMI 1640 medium, and counted by microscopy (magnification, ⫻40) using 0.4% trypan blue (Sigma Chemical Co.) to determine oncosphere viability. Oncospheres were counted using a Neubauer chamber (Fisher Scientific, Atlanta, GA). We defined nonactivated oncospheres as oncospheres that were immobile and had centrally located hooks. In contrast, we designated oncospheres activated if they showed active motility. This motility was characterized by frequent and cyclic movements of all three pairs of hooks and undulating movement of the oncosphere body. Before activated oncospheres were used in adherence assay models, they were incubated at 37°C in binding medium (RPMI 1640 medium containing 25 mM HEPES, 25 g/ml gentamicin, and 10% fetal bovine serum [pH 6.8]) for 20 min. Development of in vitro oncosphere adherence assay models. In vitro adhesion assay models were developed with three different substrates: PIMS (cells plus mucin), PSIME, and CHO-K1, Caco-2, and HCT-8 monolayer cells. Our goal was to determine which model was best suited to assessing oncosphere adherence. All of the adhesion studies were repeated three or four times on 2 or 3 separate days. The tapeworms used were from different patient sources on each day. (i) PIMS. A 10-cm portion of duodenum was collected from a pig that was 6 to 9 months old immediately postmortem and was maintained at 4°C. It was then washed with cold phosphate-buffered saline (PBS) (0.01 M dibasic sodium phosphate, 0.01 M monobasic sodium phosphate, 0.15 M NaCl; pH 7.2) to remove the intestinal contents. The small intestine was scraped, and the mucosal material (intestinal cells and mucin) was recovered and diluted in PBS. It was then fixed on eight-well slides at room temperature before oncospheres were added. Activated oncospheres were added to slides (n ⫽ 1,000) in binding medium and incubated at 37°C for 90 min. The slides were then washed in binding medium three times in order to remove oncospheres that had not bound to PIMS, were fixed in 1% glutaraldehyde, and were stained with periodic acid-Schiff stain (PAS) and hematoxylin and eosin stain (H&E). T. solium oncospheres were identified by their morphological characteristics, size, hooks, and secretory vesicles using light microscopy (magnification, ⫻100). (ii) PSIME. A 10-cm portion of duodenum was collected from pigs immediately postmortem and washed with cold PBS to remove the intestinal contents. The duodenum was placed in a tube of Earl’s balanced salts (Invitrogen, Grand Island, NY) containing penicillin (100 U/ml), streptomycin (100 g/ml), and amphotericin B (20 g/ml) and transported to the laboratory at 4°C (within 30 min). Samples were cut into full-thickness intestinal explants (approximately 0.7 by 0.7 cm) in a sterile 24-well cell culture dish. Explants were positioned with the epithelial surface up on a rectangular piece of extrathick filter paper (Bio-Rad, Hercules, CA.). This filter paper was placed in a sterile 24-well cell culture dish containing tissue culture medium consisting of Dulbecco’s modified Eagle’s medium, a high concentration of glucose (Invitrogen), and 10% inactivated fetal bovine serum to which penicillin (100 U/ml) and streptomycin (100 g/ml) were added. The level of the medium was adjusted to allow saturation of the filter
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paper, which rested on a wire mesh platform. Each piece of intestine was inoculated with 3,000 activated T. solium oncospheres in 8 l RPMI 1640 medium and incubated for 1.5 h at 37°C in an atmosphere consisting of 95% oxygen and 5% carbon dioxide. Control explants, which did not contain oncospheres, were treated in the same way. At the end of the incubation period, explants were washed three times in tissue culture medium at 37°C and then either fixed in 10% formalin at room temperature or frozen slowly in a ⫺70°C freezer. Formalin-fixed explants were examined using histochemistry to visualize oncosphere adherence to host cells by light microscopy (magnification, ⫻40). Three-micrometer sections were fixed on slides and stained with PAS and H&E. Frozen explants were sectioned and stained using immunohistochemistry by using pig polyvalent antibodies against T. solium oncospheres to visualize oncosphere adherence to host cells by UV microscopy. Polyvalent antibodies against T. solium oncospheres were obtained by subcutaneous immunization of pigs with 2 ml (0.028 mg/ml) of T. solium oncosphere extract antigens. Immunization was performed twice with a 15-day interval between the immunizations (36). The initial immunization consisted of oncosphere extract antigen emulsified in Freund’s complete adjuvant (Sigma Chemical Co.). The second immunization consisted of the same quantity of antigen emulsified in Freund’s incomplete adjuvant. Sera were collected 15 days after the second immunization as described by Verastegui et al. (35). Oncospheres that had attached to PSIME were placed in OCT compound (Sakura Finetek, Torrance, CA) and frozen, and 8-m sections were cut with a cryostat microtome. Sections on slides were then incubated with pig hyperimmune sera diluted 1:25 in PBS with 1% ovalbumin at 4°C overnight. After three washes with PBS with 1% ovalbumin, the slides were incubated for 1 h at room temperature with goat anti-pig immunoglobulin G labeled with fluorescein isothiocyanate conjugate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) diluted 1:50 in PBS with Evans blue (0.5%). The slides were then washed three times with PBS, mounted with buffered glycerin (pH 7.2), and examined by UV microscopy (magnification, ⫻100). (iii) CHO-K1 cell monolayer. Established lines of adult CHO cells (CHO-K1) were obtained from ATCC (Manassas, VA). Cultures were routinely maintained in Ham’s F-12K medium (GIBCO Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum and 40 g/ml gentamicin. Cells were incubated under 5% CO2 in air at 37°C. The medium was changed every 2 days, and cells were passaged when a monolayer reached confluence. CHO cells were harvested using trypsin-EDTA treatment (Sigma Chemical Co.) after a complete monolayer was formed in a culture flask. The cells were then seeded into an eight-well chamber slide system with 0.7-cm2 wells (Nalgene-Nunc). The T. solium oncosphere adherence assay was performed using confluent CHO cell monolayers in eight-well chamber slides, as previously described (6, 21, 29, 32). (a) Comparison of T. solium oncosphere adherence to viable and fixed CHO-K1 cells. Viable monolayer cells were used immediately after monolayer formation in a chamber slide. The number of oncospheres adhering to viable monolayer CHO-K1 cells was compared to the number of oncospheres adhering to fixed monolayer CHO-K1 cells after 48 h of cell culture. The fixed monolayer CHO cells were treated with 1% formaldehyde for 1 h and subsequently stored in PBS at 4°C until they were used (34). Viable and fixed monolayer cells were incubated with 2,500 T. solium activated oncospheres in binding medium at 37°C for 1.5 h. After incubation, the unbound oncospheres were rinsed three times with binding medium, and the oncospheres bound to the cells were fixed with 1% glutaraldehyde in PBS and stained with PAS. Adherent oncospheres were then counted by light microscopy (magnification, ⫻100). (b) Standardization of optimum time of oncosphere incubation for adherence. Forty-eight-hour viable monolayer CHO cells maintained at 37°C with 5% CO2 were incubated with 2,500 activated oncospheres in binding medium for different times (30 min and 1.5, 3, and 24 h) to determine the optimum time of oncosphere incubation to obtain the highest number of adherent oncospheres. (c) Role of sera and cell maturation in oncosphere adherence. The effect of sera on oncosphere adherence in the CHO cell model was examined. Viable monolayer CHO-K1 cells were tested at different times during monolayer maturation (24, 48, and 72 h) in the chamber slide system. Monolayer cells were incubated with 2,500 activated oncospheres in RPMI medium both with and without 10% fetal bovine serum (RPMI 1640 containing 25 mM HEPES and 25 g/ml of gentamicin, pH 6.8) at 37°C with 5% CO2. Oncosphere adhesion was evaluated after 30 min and 1 h of incubation. The effect of varying the fetal bovine serum concentration (1, 2.5, 5, 7.5, 10, 15, and 20%) on oncosphere adherence was also evaluated using viable monolayer CHO-K1 cells. The number of oncospheres bound to monolayer cells was determined by PAS staining and light microscopy (magnification, ⫻100). (d) Effect of temperature on oncosphere adherence. The effect of temperature on oncosphere adhesion to the CHO cells was examined at different incubation temperatures. Viable monolayer cells were incubated with 2,500 T. solium acti-
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FIG. 1. (Panel i) T. solium oncospheres adhering to PIMS. Oncosphere adhesion was visualized by light microscopy using PAS. Magnification, ⫻40. Arrows A, oncospheres adhering to PIMS; arrows B, oncosphere hooks. (Panel ii) T. solium oncosphere binding to viable monolayer CHO cells. The arrows indicate secretory vesicles. Magnification, ⫻40. (Panel iii) T. solium oncosphere binding to fixed monolayer CHO cells visualized using PAS. The arrows indicate one pair of penetration glands. Magnification, ⫻100. (Panel iv) Light microscopy of 3-m porcine small intestinal section embedded in paraffin and stained with H&E (20-min incubation period). An oncosphere (arrow) is interacting with epithelial cells. Magnification, ⫻400. (Panels v and vi) Photographs of frozen 8-m sections, obtained using light and UV microscopy, showing oncospheres (arrows) identified by the presence of hooks and immunofluorescence (20-min incubation period; magnification, ⫻100). A light microscopy photograph with Evans blue (panel v) and a UV microscopy photograph (panel vi) show the same field of view. Material stained with a contrast agent appears to be blue under visible light and red under UV light, while fluorescence is green under UV light.
vated oncospheres in binding medium at 4, 12, 24, and 37°C for 30 min and 1 h. After incubation, slides were rinsed three times with binding medium to remove unbound oncospheres. Oncospheres bound to the cells were fixed with 1% glutaraldehyde in PBS and stained with PAS. (e) Scanning electron microscopy (SEM). Activated T. solium oncospheres that were adherent to formalin-fixed monolayer CHO cells were incubated for 2 h with 1% glutaraldehyde in PBS (pH 7.2) plus 1% sucrose. The specimens were then washed three times with 1% glutaraldehyde in PBS (pH 7.2) plus 1% sucrose and stored at 4°C until they were used. The preparations
were postfixed in 1.5% osmium tetroxide, and serial dehydration was performed using 70, 95, and 100% ethanol (2 h each). Samples were then processed in a critical point bomb using liquid carbon dioxide as a transitional fluid and then sputter coated using a Denton S-II instrument with a goldpalladium target. Images were obtained using a Hitachi 2400 microscope at 15 kV. (f) Transmission electron microscopy. Pellets of activated oncospheres and activated oncospheres adhering to PSIME were processed as described above for SEM, and then samples were postfixed in 1.5% osmium tetroxide, dehydrated in
FIG. 2. Ninety-five percent confidence intervals for oncospheres adherent on unfixed monolayer CHO cells after 30 min and 1.5, 3, and 25 h of incubation at 37°C.
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FIG. 3. (a) Ninety-five percent confidence intervals for the number of oncospheres adhering on unfixed CHO-K1 cells after 1.5 h of incubation at 37°C with binding medium with serum and binding medium without serum (RPMI), obtained using 24-, 48-, and 72-h mature monolayer CHO cells. (b) Curve and 95% confidence intervals for the number of oncospheres adhering on unfixed CHO-K1 cells after 1.5 h of incubation at 37°C with binding medium with different fetal bovine serum concentrations (0, 1, 2.5, 5, 7.5, 10, 15, and 20%), determined with 48-h mature monolayer CHO-K1 cells.
alcohol, and embedded in epoxy, and 50-nm sections were cut. Sections were then stained serially with lead hydroxide and uranyl acetate and were examined using a Phillips electron microscope (Phillips Electronic Instruments, Eindhoven, The Netherlands) operating at 75 kV. (iv) Detection of oncosphere interaction with microvilli by immunofluorescence. Established cell lines HCT-8, Caco-2, and CHO were used to determine the presence of microvilli in adherent oncospheres. CHO, HCT-8, and Caco-2 cells were obtained from ATCC (Manassas, VA). CHO cells were cultured as described above. HCT-8 cells were cultured in RPMI 1640 medium with 10% equine serum. Caco-2 cell cultures were routinely maintained in Eagle minimum essential medium with 20% fetal bovine serum. HCT-8, Caco-2, and CHO cells were incubated under 5% CO2 in air at 37°C. The HCT-8, Caco-2, and CHO cells were harvested using trypsin-EDTA treatment (Sigma Chemical Co.) after a complete cell monolayer was formed in a culture flask. The cells were then seeded into an eight-well chamber slide system with 0.7-cm2 wells (NalgeneNunc). The T. solium oncosphere adherence assay was performed using confluent CHO, HCT-8, and Caco-2 cell monolayers in eight-well chamber slides, as described above. Viable monolayer cells were incubated with 3,000 T. solium activated oncospheres in binding medium at 37°C for 1.5 h. After incubation, unbound oncospheres were rinsed three times with binding medium, and the
oncospheres bound to the cells were fixed with 50% acetone and methanol and stained by immunofluorescence using rabbit polyvalent antibodies against T. solium oncospheres to visualize oncosphere adherence to monolayer cells using UV microscopy (magnification, ⫻40). Polyvalent antibodies against T. solium oncospheres were obtained by subcutaneous immunization of rabbits with 2 ml (0.028 mg/ml) of T. solium activated oncosphere extract antigens. Immunization was performed four times, with the second immunization 15 days after the first immunization and with the subsequent immunizations at 1-week intervals. The initial immunization was with oncosphere extract antigen emulsified in Freund’s complete adjuvant (Sigma Chemical Co.). The subsequent immunizations were with the same quantity of antigen emulsified in Freund’s incomplete adjuvant. Sera were collected 15 days after the fourth immunization. Slides containing fixed monolayer cells with adherent oncospheres were then incubated with rabbit hyperimmune sera diluted 1:100 in PBS with 1% ovalbumin and 0.05% Tween 20 (PBS-TO) at room temperature for 1 h. After three washes with PBS-TO, the slides were incubated for 1 h at room temperature with goat anti-rabbit immunoglobulin G labeled with fluorescein isothiocyanate conjugate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) diluted 1:100 in PBS-TO plus Evans blue (0.5%). The slides were then washed three times with
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FIG. 4. Curve and 95% confidence intervals for the number of oncospheres adhering on unfixed CHO-K1 cells after 1.5 h of incubation at different temperatures.
PBS, mounted with buffered glycerin (pH 7.2), and examined by UV microscopy (magnification, ⫻100). Measurement of T. solium oncosphere adherence in each model. In order to allow comparisons between models, the proportion of adherent oncospheres was estimated by dividing the number of oncospheres adhering to cells by the total number of oncospheres added to the monolayer surface. Means, distributions, and confidence limits (␣ ⫽ 0.05) of adherent oncospheres were estimated for each model.
RESULTS Development of in vitro oncosphere adherence assay model. (i) PIMS. The percentage of oncosphere activation was between 50 and 75% after incubation in sodium hypochlorite and artificial intestinal fluids. After treatment with the artificial intestinal fluids, the hooks protruded from the cell and the oncosphere membrane was shed. An activated oncosphere was characterized by its motion and movement of its hooks; some hooks projected outside
the oncosphere (Fig. 1, panel i). Also, single or multiple oncosphere secretory vesicles appeared outside the oncosphere adjacent to the oncosphere surface (Fig. 1, panel ii). The overall percentage of oncospheres adhering to PIMS ranged from 2 to 37%. In this model, it was important to have a good-quality stain to identify the oncospheres on pig intestinal mucosa. PAS gave better resolution than H&E, and identification of the hooks and other oncosphere morphological characteristics was easier (Fig. 1, panels i and iii). (ii) PSIME. Initially, we assessed porcine small intestine tissue culture survival time. The histological cellular appearance of the epithelium and subepithelial tissue throughout this period was relatively normal up to a culture time of 4.5 h; after this cell degeneration occurred. Thus, oncosphere adherence studies were not done after 4.5 h of culture. When activated oncospheres were incubated with explants, a
FIG. 5. Scanning electron micrographs of T. solium oncospheres adhering to fixed monolayer CHO-K1 cells. (a) T. solium oncospheres attached to the surface of CHO cells by elongated microvilli. (b) Secretory vesicles (arrows) that are present outside the oncosphere membrane. Microvilli are on the surface membrane of the oncosphere and secretory vesicles. (c) Elongate microvilli that are attached to the surface of CHO cells. Hooks are present outside the oncosphere.
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number of oncosphere forms (up to 10) were observed adhering to the epithelial lining of the intestine following histological sectioning of both formalin-fixed explants (Fig. 1 panel iv) and frozen explants (Fig. 1, panels v and vi). The appearance of sections and oncospheres was demonstrated by H&E staining (Fig. 1, panel iv). In frozen sections treated with anti-T. solium oncosphere antibodies, both oncospheres and the surrounding material were visualized by immunofluorescence (Fig. 1, panel vi). Adding Evans blue stain to the conjugate material permitted visualization of oncosphere hooks within the oncospheres (Fig. 1, panel v). (iii) Monolayer CHO-K1 cells. (a) T. solium oncosphere adherence to viable and fixed CHO-K1 cells. Activated oncospheres adhered to both viable and fixed monolayer CHO cells (Fig. 1, panels ii and iii). The number of oncospheres bound to viable monolayer CHO cells was higher than the number bound to fixed monolayer cells (means, 386 ⫾ 102 and 78 ⫾ 57 oncospheres, respectively; P ⬍ 0.05). Oncosphere activation was important for adhesion to monolayer cells since 95% of adherent oncospheres were activated. (b) Standardization of optimum time of oncosphere incubation for adherence. Incubation for 1.5 h yielded the highest number of adherent oncospheres on viable monolayer CHO cells (Fig. 2). (c) Role of serum and cell maturation in oncosphere adherence. As Fig. 3a shows, the use of binding medium with 10% fetal bovine serum resulted in a significantly greater number of adhering oncospheres than the use of binding medium without serum (P ⬍ 0.05) at all times during monolayer cell maturation. When binding medium with serum was used, the optimum time of monolayer cell maturation was 48 h. The adherence of oncospheres increased linearly as the serum concentration in the binding medium increased from 1 to 5% and then plateaued (Fig. 3b). (d) Effect of temperature on oncosphere adherence. The number of oncospheres adhering to viable monolayer cells increased when the incubation temperature was increased from 12 to 37°C (Fig. 4). The numbers of adherent oncospheres were statistically significantly different at 12, 24, and 37°C (P ⬍ 0.05) (Fig. 4). The adherent oncospheres had visible secretory vesicles, and the numbers of these vesicles increased as the incubation temperature increased. Secretory vesicles (Fig. 5b) were seen in about 80 to 85% of adherent oncospheres incubated at 37°C but in only 10% of adherent oncospheres incubated at 12 or 4°C. The sizes of the secretory vesicles varied from 2.07 to 3.71 m (median, 2.65 m). (e) SEM. SEM showed that the surface membrane or epithelium of oncospheres adhering to CHO cells had elongated microvilli. The elongated microvilli extended and appeared to attach to the surface of the monolayer cells (Fig. 5a, 5b, and 5c). Also, these microvilli were present on the secretory vesicle membrane, similar to surface membranes of the oncosphere (Fig. 5b). The secretory vesicles appeared to be attached to both the CHO cells and the oncospheres via their elongated microvilli. (f) Transmission electron microscopy. Transmission electron microscopy showed that the first microvilli were formed under the oncosphere membrane of the activated oncosphere
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FIG. 6. (a) Transmission electron microscopy of activated T. solium oncosphere. The arrows indicate microvilli (MV) that are under the oncosphere membrane (OM). (b) Transmission electron microscopy of activated T. solium oncosphere (Onc) adhering to pig small intestinal cell (IC). The large arrow indicates the microvilli (MV).
(Fig. 6a). Microvilli appeared to attach to the pig intestinal cells using the PSIME model (Fig. 6b). (iv) Detection of oncosphere microvilli by immunofluorescence. As determined by UV microscopy, the surface membranes of oncospheres adhering to CHO, HCT-8, and Caco-2 cells all reacted strongly with hyperimmune rabbit sera against
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20 µ m
20 µ m
a
b
20 µ m
c
FIG. 7. UV microscopy (magnification, ⫻100) of T. solium oncospheres adhering to viable monolayer CHO cells. (a and b) Activated T. solium oncospheres without oncosphere membrane attached to the surface of CHO cells by elongated microvilli. The arrows indicate the elongated microvilli and secretory vesicles that are present outside the oncosphere membrane. (c) Activated T. solium oncosphere with oncosphere membrane adhering to a viable monolayer CHO cell.
T. solium activated oncospheres (Fig. 7). Oncospheres, when tested with each cell line, had elongated microvilli and secretory vesicles (Fig. 7a and 7b). Also, it was possible to differentiate between activated oncospheres with an oncosphere membrane and activated oncospheres without an oncosphere membrane (Fig. 7c). Microvillar fragments adhering to monolayer cells were seen ⬎50 m from the oncosphere surface (Fig. 7a). DISCUSSION The present study showed that adherence of activated T. solium oncospheres can be observed in vitro and can be measured objectively using the following three different substrates: PIMS (cells plus mucin), PSIME, and monolayer cells (CHOK1, Caco-2, and HCT-8 cells). The reproducibility of T. solium oncosphere adherence was most easily measured with CHO cells. Serum and an increased incubation temperature enhanced adherence. Our studies also demonstrated that surface membranes of adherent T. solium oncospheres had elongated microvilli that attached to tissue cultures and expelled external secretory vesicles with the same elongated microvilli. These elongated microvilli appeared to attach to the CHO cells. The PIMS model was used because the mechanism of oncosphere adherence resembled natural infection as it occurs on the porcine small intestinal mucosal surface. This model has the advantage that fresh tissue is not needed for each experiment. Rather, the mucosal material can be fixed on slides and used for multiple studies. The PIMS model includes both intestinal epithelial cells and mucin. In this model, the mucin and the epithelial cells may have different receptors that cannot be distinguished. Also, the detection of oncospheres by staining is somewhat difficult due to high background staining. The PSIME model resembles the natural infection that occurs in pigs, but it has the disadvantage that it requires fresh porcine small intestinal mucosa for each experiment. Nonetheless, the PSIME model is useful for determining immunohistological characteristics of early host-oncosphere interactions.
The third model, using monolayer CHO cells, is valuable since this cell line has glycosylated mutants, which can be used in future studies to characterize the oncosphere lectin or cell carbohydrate receptors that are involved in the adherence mechanisms. CHO cells have been employed to investigate attachment of other parasites, including E. histolytica and Trichomonas (6, 28). However, monolayer CHO-K1 cells may not accurately mimic the natural process of adherence. T. solium oncosphere adhesion to monolayer CHO cells was enhanced by the presence of serum in binding medium, indicating that unidentified serum factors play a primary role in the oncosphere-host cell interaction. Similarly, the adherence of Echinococcus multilocularis and Echinococcus granulosus protoscoleces to human endothelial cell monolayers was enhanced by sera (19). One of the serum factors known to play a leading role in cell-cell and cell-substratum interactions is fibronectin (27). It has been shown that fibronectin accumulates in areas of cell-cell contact and promotes the adhesion between certain tissues and cells (for example, fibroblasts and collagen substratum) (8, 26). In addition, there are other known or putative modulators of adhesion, such as proteoglycans, various collagen types, laminin, and vitronectin, that require further examination (30). The movement and morphology of activated T. solium oncospheres are similar to the movement and morphology described previously for other cestodes (5, 23, 25, 31). This study demonstrates that secretory vesicles are expelled outside the activated T. solium oncospheres upon activation. These secretory vesicles may be important either for attachment to host tissues, to facilitate penetration through the epithelium, or to help protect the oncospheres against digestive enzymes. However, these secretory vesicles have been found previously within oncospheres (16, 31). Our study is the first study showing secretory vesicles outside the oncosphere body. Secretory vesicles can be seen outside oncospheres with and without the presence of cells; however, activation of the oncospheres is required. The formation of secretory vesicles that we observed may be
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somewhat similar to that reported for Taenia saginata, in which, after oncosphere activation, changes in the composition of the cell cytoplasm of penetration glands can be observed by electron microscopy. When the oncosphere membrane was eliminated, the vesicles could be seen on the surface of the oncosphere (31). Other authors have mentioned that the secretion of these vesicles is initiated and maintained by the muscular contractions of the oncosphere (9). Our findings, however, differ from those of previous reports since we did not find the secretory vesicles separated from the oncosphere as part of the maturation process. Microvilli were previously described for E. granulosus (16, 17), Taenia taeniaeformis (11), T. saginata (31), and Taenia ovis oncospheres (15) using transmission microscopy. However, in the presence of CHO, HCT-8, and Caco-2 cells, the T. solium oncospheres developed elongated microvilli that attached to the tissue culture cells. These T. solium oncospheres with elongated microvilli appear to have been described previously by Engelkirk and Williams but without reference to their function (11). These authors noted that in rats infected orally with T. taeniaformis eggs, oncospheres were present in the liver 24 h postinfection, with long microvilli that appear to be similar to what we described. In 1987, Harris et al. also showed that after 48 h of in vitro incubation in tissue culture T. ovis oncospheres had elongated microvilli (15). Our model of T. solium oncosphere infection in tissue culture differs in that microvilli have been found in association with both the oncospheres and their secretory vesicles. In addition, our model strongly supports the notion that elongated microvilli are involved in the adherence of oncospheres to host cells. What remains to be determined is the precise timing of the formation of the microvilli (i.e., upon stimuli received from host cells or upon direct contact). Elongated microvilli were shown to have a strong reaction with hyperimmune sera against oncospheres, showing that microvillus antigens are more immunogenic. Inhibition of this adherence mechanism may provide a useful method to block T. solium infection. Also still to be resolved is the precise role(s) of the secretory vesicles. Do they play a significant role in allowing the oncosphere access into and possibly through host intestinal tissues? This study demonstrated three models for T. solium oncosphere adherence. It also demonstrated that immunofluorescence is a useful tool for observing differentially activated oncospheres with and without oncosphere membranes. The CHO cell model has the advantage of being easily modified and is able to assess different individual variables important for adherence. This model should now permit us to characterize in more detail the mechanism of T. solium oncosphere adherence.
ACKNOWLEDGMENTS Funding for this project came from Gates project 23981, NIH Global Research training grant D43 TW006581, Wellcome Trust grant GR063109MA, and the RG-ER Anonymous Fund for Tropical Research. We thank Charles Sterling, Paula Maguin ˜a for administrative help, and J. B. Phu, D. Sara, and Sari-CeCe for technical assistance.
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