Centre for Medical Parasitology, Institute for Medical Microbiology and Immunology and Copenhagen University Hospital (Rigshospitalet), Univ
http://www.100md.com
感染与免疫杂志 2006年第5期
Centre for Medical Parasitology, Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet) and Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark
Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana
ABSTRACT
Placenta-sequestering Plasmodium falciparum parasites causing pregnancy-associated malaria express pregnancy-specific variant surface antigens (VSAPAM). We report here that VSAPAM-expressing patient isolates adhere strongly to the choriocarcinoma cell line BeWo and that the BeWo line can be used to efficiently select for VSAPAM expression in vitro.
TEXT
Women living in areas of stable Plasmodium falciparum transmission become highly susceptible to infection during their first pregnancy, regardless of previously acquired protective immunity (reviewed in reference 7). The resulting pregnancy-associated malaria (PAM), which is caused by parasites selectively accumulating in the placenta, is an important cause of poor mother-child health in areas where P. falciparum is endemic (18). Placenta-sequestering infected erythrocytes (IE) express particular parasite-encoded variant surface antigens (VSA) on the IE surface that are functionally and antigenically distinct from VSA expressed by nonplacental IE (1, 6, 13). Functionally, the PAM-specific VSA (VSAPAM) are unique in mediating IE adhesion to placental host receptors such as the glycosaminoglycan chondroitin sulfate A (CSA) that are not receptors for VSA expressed by IE in nonpregnant individuals. The fact that P. falciparum-exposed males and females who have never been pregnant do not possess VSAPAM-specific antibodies, despite high levels of antibodies specific for other VSA, is evidence of the antigenic distinctiveness of VSAPAM. These findings, and the evidence that links VSAPAM-specific immunoglobulin G (IgG) to acquired immunological protection from the adverse clinical consequences of PAM (5, 17), suggest that VSAPAM can be used in a syndrome-specific vaccine against PAM. Consequently, functional and molecular characterization of VSAPAM and, not least, determination of the intraclonal and interclonal diversity of these antigens are a current high-priority research area (14).
Some P. falciparum lines can acquire the VSAPAM phenotype following in vitro selection for IE adhesion to CSA (13, 16), but this type of selection is inefficient with other lines (our unpublished data). Furthermore, many placental P. falciparum isolates have relatively low affinity for CSA (6), while some nonplacental isolates have been reported to adhere to CSA (3). Finally, recent studies have shown that IE can acquire the ability to adhere to the placental choriocarcinoma cell line BeWo following selection in vitro and that this adhesion can be partially inhibited by soluble CSA and chondroitinase treatment (9, 20). These studies suggest that IE adhesion to BeWo cells may be a valuable tool in the characterization of the adhesion specificity of VSAPAM-expressing parasites isolated from PAM patients and that this cell line can be used for efficient selection of VSAPAM-expressing IE in vitro. However, no patient isolates were included in the earlier studies (9, 20), and other than line 3D7 only long-term in vitro adapted laboratory lines that can be readily selected for IE binding to CSA were used. Finally, acquisition of VSAPAM expression in response to selection of IE for adhesion to BeWo cells in vitro has not previously been documented.
To further validate the role of BeWo cells in PAM research, we first examined the VSA expression of 16 P. falciparum patient isolates obtained from the peripheral blood of pregnant women living in an area of stable parasite transmission. The isolates were collected, and their VSA expression was characterized as previously described (12). Fifteen of the isolates clearly expressed VSAPAM when tested in a flow cytometry assay (13, 15). Thus, for each of these isolates the levels of VSA-specific IgG in the plasma of 27 P. falciparum-exposed multiparous women were much higher than levels in plasma from 30 sympatric men (t test, P < 1 x 10–7 for each isolate) (Fig. 1). This strongly sex-specific recognition implies a placental infection focus (12). Plasma levels of IgG with specificity for the VSA expressed by isolate DP168 were less sex specific (t test, P > 5 x 10–3) (Fig. 1). Thus, the VSA expressed by this isolate appeared to be a mixture of VSAPAM and non-PAM VSA (12).
We next measured the ability of IE from the 16 patient isolates to adhere to CSA and to four cell lines purchased from American Type Culture Collection (http://www.lgcpromochem-atcc.com). The lines used were the Chinese hamster ovary (CHO) line K1 (ATCC CCL-61) that expresses CSA, a CHO glycosylation mutant line (A-745; ATCC CRL-2242) that does not express CSA, a CHO cell line transfected with human CD36 (CHO-CD36; ATCC CRL-2092), and the choriocarcinoma cell line BeWo (ATCC CCL-98). All cell lines were maintained as suggested by the American Type Culture Collection. In some preliminary experiments, we pretreated the BeWo cells with forskolin to induce multinucleated syncytia (23). However, forskolin treatment did not markedly affect selection efficiency, and forskolin was not used in the results presented in this paper.
Compared to the CSA-deficient cell line (A-745), the 15 VSAPAM-expressing P. falciparum patient isolates bound significantly better to CSA and to each of the other cell lines (Kruskal-Wallis test, P < 0.001, and Dunn's post hoc test, P < 0.05 in all cases) (Fig. 2). Adhesion of these isolates to CSA and to the CSA-expressing CHO cell lines (CHO-K1 and CHO-CD36) was similar, whereas adhesion to the BeWo line was significantly higher (Kruskal-Wallis test, P < 0.001, and Dunn's post hoc test, P < 0.05 CSA versus BeWo) (Fig. 2). DP168 did not bind well to CSA but bound strongly to CHO-CD36 (Fig. 2). A subline of the long-term in vitro-adapted P. falciparum line FCR3 (FCR3-CHO) that expressed VSAPAM (t test as above, P < 1 x 10–10) following selection for binding to K1 and included as a control for CSA-mediated adhesion (10) bound well to CSA and to all the CSA-expressing lines (Fig. 2). In contrast, a CD36-adhering FCR3 subline (FCR3-CD36) expressing non-PAM VSA (t test as above, P = 0.16) and included as a control line for CD36-mediated binding (10) only bound to the CD36-transfected CHO cell line (Fig. 2). Specifically, FRC3-CD36 did not bind well to the BeWo cell line, which does not express CD36 (20). These results show that VSAPAM-expressing patient isolates bind strongly to BeWo cells, suggesting that this line is a useful tool in selecting for VSAPAM-expression in vitro.
To test this hypothesis, we subjected six long-term in vitro adapted laboratory lines (3D7, Dd2, FCR3, HB-3, K1, and NF54) to selection for adhesion to BeWo cells (Table 1). The lines were chosen because none of them spontaneously expressed VSAPAM and because our attempts to select for VSAPAM expression by 10 rounds of panning on CSA (13) had been unsuccessful for four of them (Table 1). For three of five lines tested, selection for VSAPAM expression by selection for adhesion to the K1 cell line (10) had been equally ineffective (Table 1). After only three rounds of selection for adhesion to BeWo cells, three of the lines had clearly acquired VSAPAM expression (Table 1 and Fig. 3). With respect to 3D7, plasma levels of VSA-specific IgG were lower (paired t test, P = 0.01) in males and higher in multiparous women (P = 0.0001) after three rounds of selection, but substantial male reactivity remained, indicating that acquisition of VSAPAM was incomplete for this clone.
Finally, we subjected three P. falciparum isolates (Busua, N0031, and N0045) recently obtained from nonpregnant malaria patients to selection for IE adhesion to BeWo cells. Two of these isolates rapidly acquired VSAPAM expression (Table 1). Although the VSA expressed by the third isolate clearly changed in response to selection, it did not become VSAPAM-like, and the IE from this isolate appeared to have affinity for the plastic surface of culture plates rather than for receptors on the BeWo cells (data not shown).
In conclusion, we have shown that VSAPAM-expressing P. falciparum patient isolates implicated in the pathogenesis of PAM adhere more strongly to the placental choriocarcinoma cell line BeWo than to CSA and cell lines often used in studies of P. falciparum involved in PAM. Furthermore, we demonstrate that the BeWo cell line is an efficient tool to select for VSAPAM expression in vitro. These findings establish the BeWo cell line as an important tool for research on the VSAPAM that are centrally involved in the pathogenesis of and protective immunity to PAM. Studies on the ability of VSAPAM-specific IgG to interfere with adhesion of VSAPAM-expressing IE are currently under way.
ACKNOWLEDGMENTS
We are indebted to all the individuals who donated parasite and plasma samples for the study. Kirsten Pihl and Maiken Christensen are thanked for excellent technical assistance in the laboratory.
The work received financial support from the Commission of European Union (grant QLK2-CT-2001-01302, PAMVAC), from the Danish Medical Research Council (SSVF; grants 22-02-0571 and 22-03-0333), and from the Danish Research Council for Development Research (RUF; grant 104.Dan.8.L.306). R.H. was supported by a scholarship from SSVF (grant 22-04-0012).
REFERENCES
1. Beeson, J. G., G. V. Brown, M. E. Molyneux, C. Mhango, F. Dzinjalamala, and S. J. Rogerson. 1999. Plasmodium falciparum isolates from infected pregnant women and children are associated with distinct adhesive and antigenic properties. J. Infect. Dis. 180:464-472.
2. Bhasin, V. K., and W. Trager. 1984. Gametocyte-forming and non-gametocyte-forming clones of Plasmodium falciparum. Am. J. Trop. Med. Hyg. 33:534-537.
3. Chaiyaroj, S. C., P. Angkasekwinai, A. Buranakiti, S. Looareesuwan, S. J. Rogerson, and G. V. Brown. 1996. Cytoadherence characteristics of Plasmodium falciparum isolates from Thailand: evidence for chondroitin sulfate A as a cytoadherence receptor. Am. J. Trop. Med. Hyg. 55:76-80.
4. Delemarre, B. J., and H. J. Van der Kaay. 1979. Malaria tropica op natuurlijke wijze verkregen in Nederland. Ned. Tijdschr. Geneeskd. 123:1981-1982.
5. Duffy, P. E., and M. Fried. 2003. Antibodies that inhibit Plasmodium falciparum adhesion to chondroitin sulfate A are associated with increased birth weight and the gestational age of newborns. Infect. Immun. 71:6620-6623.
6. Fried, M., and P. E. Duffy. 1996. Adherence of Plasmodium falciparum to chondroitin sulfate A in the human placenta. Science 272:1502-1504.
7. Hviid, L. 2004. The immuno-epidemiology of pregnancy-associated malaria: a variant surface antigen-specific perspective. Parasite Immunol. 26:477-486.
8. Jensen, J. B., and W. Trager. 1978. Plasmodium falciparum in culture: establishment of additional strains. Am. J. Trop. Med. Hyg. 27:743-746.
9. Lucchi, N. W., R. Koopman, D. S. Peterson, and J. M. Moore. Plasmodium falciparum-infected red blood cells selected for binding to cultured syncytiotrophoblast bind to chondroitin sulfate A and induce tyrosine phosphorylation in the syncytiotrophoblast. Placenta, in press.
10. Megnekou, R., T. Staalsoe, D. W. Taylor, R. Leke, and L. Hviid. 2005. Effects of pregnancy and intensity of Plasmodium falciparum transmission on immunoglobulin G subclass responses to variant surface antigens. Infect. Immun. 73:4112-4118.
11. Oduola, A. M., W. K. Milhous, N. F. Weatherly, J. H. Bowdre, and R. E. Desjardins. 1988. Plasmodium falciparum: induction of resistance to mefloquine in cloned strains by continuous drug exposure in vitro. Exp. Parasitol. 67:354-360.
12. Ofori, M. F., T. Staalsoe, V. Bam, M. Lundquist, K. P. David, E. N. L. Browne, B. D. Akanmori, and L. Hviid. 2003. Expression of variant surface antigens by Plasmodium falciparum parasites in the peripheral blood of clinically immune pregnant women indicates ongoing placental infection. Infect. Immun. 71:1584-1586.
13. Ricke, C. H., T. Staalsoe, K. Koram, B. D. Akanmori, E. M. Riley, T. G. Theander, and L. Hviid. 2000. Plasma antibodies from malaria-exposed pregnant women recognize variant surface antigens on Plasmodium falciparum-infected erythrocytes in a parity-dependent manner and block parasite adhesion to chondroitin sulfate A. J. Immunol. 165:3309-3316.
14. Smith, J. D., and K. W. Deitsch. 2004. Pregnancy-associated malaria and the prospects for syndrome-specific antimalaria vaccines. J. Exp. Med. 200:1093-1097.
15. Staalsoe, T., H. A. Giha, D. Dodoo, T. G. Theander, and L. Hviid. 1999. Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry. Cytometry 35:329-336.
16. Staalsoe, T., R. Megnekou, N. Fievet, C. H. Ricke, H. D. Zornig, R. Leke, D. W. Taylor, P. Deloron, and L. Hviid. 2001. Acquisition and decay of antibodies to pregnancy-associated variant antigens on the surface of Plasmodium falciparum-infected erythrocytes that are associated with protection against placental parasitemia. J. Infect. Dis. 184:618-626.
17. Staalsoe, T., C. E. Shulman, J. N. Bulmer, K. Kawuondo, K. Marsh, and L. Hviid. 2004. Variant surface antigen-specific IgG and protection against the clinical consequences of pregnancy-associated Plasmodium falciparum malaria. Lancet 363:283-289.
18. Steketee, R. W., B. L. Nahlen, M. E. Parise, and C. Menendez. 2001. The burden of malaria in pregnancy in malaria-endemic areas. Am. J. Trop. Med. Hyg. 64:28-35.
19. Thaithong, S., and G. H. Beale. 1981. Resistance of ten Thai isolates of Plasmodium falciparum to chloroquine and pyrimethamine by in vitro tests. Trans. R. Soc. Trop. Med. Hyg. 75:271-273.
20. Viebig, N. K., M. C. Nunes, A. Scherf, and B. Gamain. 2006. The human placental derived BeWo cell line: a useful model for selecting Plasmodium falciparum CSA-binding parasites. Exp. Parasitol. 112:121-125.
21. Walliker, D., I. A. Quakyi, T. E. Wellems, T. F. McCutchan, A. Szarfman, W. T. London, L. M. Corcoran, T. R. Burkot, and R. Carter. 1987. Genetic analysis of the human malaria parasite Plasmodium falciparum. Science 236:1661-1666.
22. Wellems, T. E., L. J. Panton, I. Y. Gluzman, R. Do, V., R. W. Gwadz, A. Walker-Jonah, and D. J. Krogstad. 1990. Chloroquine resistance not linked to mdr-like genes in a Plasmodium falciparum cross. Nature 345:253-255.
23. Wice, B., D. Menton, H. Geuze, and A. L. Schwartz. 1990. Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation in vitro. Exp. Cell Res. 186:306-316.(Rikke N. Haase, Rosette M)
Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana
ABSTRACT
Placenta-sequestering Plasmodium falciparum parasites causing pregnancy-associated malaria express pregnancy-specific variant surface antigens (VSAPAM). We report here that VSAPAM-expressing patient isolates adhere strongly to the choriocarcinoma cell line BeWo and that the BeWo line can be used to efficiently select for VSAPAM expression in vitro.
TEXT
Women living in areas of stable Plasmodium falciparum transmission become highly susceptible to infection during their first pregnancy, regardless of previously acquired protective immunity (reviewed in reference 7). The resulting pregnancy-associated malaria (PAM), which is caused by parasites selectively accumulating in the placenta, is an important cause of poor mother-child health in areas where P. falciparum is endemic (18). Placenta-sequestering infected erythrocytes (IE) express particular parasite-encoded variant surface antigens (VSA) on the IE surface that are functionally and antigenically distinct from VSA expressed by nonplacental IE (1, 6, 13). Functionally, the PAM-specific VSA (VSAPAM) are unique in mediating IE adhesion to placental host receptors such as the glycosaminoglycan chondroitin sulfate A (CSA) that are not receptors for VSA expressed by IE in nonpregnant individuals. The fact that P. falciparum-exposed males and females who have never been pregnant do not possess VSAPAM-specific antibodies, despite high levels of antibodies specific for other VSA, is evidence of the antigenic distinctiveness of VSAPAM. These findings, and the evidence that links VSAPAM-specific immunoglobulin G (IgG) to acquired immunological protection from the adverse clinical consequences of PAM (5, 17), suggest that VSAPAM can be used in a syndrome-specific vaccine against PAM. Consequently, functional and molecular characterization of VSAPAM and, not least, determination of the intraclonal and interclonal diversity of these antigens are a current high-priority research area (14).
Some P. falciparum lines can acquire the VSAPAM phenotype following in vitro selection for IE adhesion to CSA (13, 16), but this type of selection is inefficient with other lines (our unpublished data). Furthermore, many placental P. falciparum isolates have relatively low affinity for CSA (6), while some nonplacental isolates have been reported to adhere to CSA (3). Finally, recent studies have shown that IE can acquire the ability to adhere to the placental choriocarcinoma cell line BeWo following selection in vitro and that this adhesion can be partially inhibited by soluble CSA and chondroitinase treatment (9, 20). These studies suggest that IE adhesion to BeWo cells may be a valuable tool in the characterization of the adhesion specificity of VSAPAM-expressing parasites isolated from PAM patients and that this cell line can be used for efficient selection of VSAPAM-expressing IE in vitro. However, no patient isolates were included in the earlier studies (9, 20), and other than line 3D7 only long-term in vitro adapted laboratory lines that can be readily selected for IE binding to CSA were used. Finally, acquisition of VSAPAM expression in response to selection of IE for adhesion to BeWo cells in vitro has not previously been documented.
To further validate the role of BeWo cells in PAM research, we first examined the VSA expression of 16 P. falciparum patient isolates obtained from the peripheral blood of pregnant women living in an area of stable parasite transmission. The isolates were collected, and their VSA expression was characterized as previously described (12). Fifteen of the isolates clearly expressed VSAPAM when tested in a flow cytometry assay (13, 15). Thus, for each of these isolates the levels of VSA-specific IgG in the plasma of 27 P. falciparum-exposed multiparous women were much higher than levels in plasma from 30 sympatric men (t test, P < 1 x 10–7 for each isolate) (Fig. 1). This strongly sex-specific recognition implies a placental infection focus (12). Plasma levels of IgG with specificity for the VSA expressed by isolate DP168 were less sex specific (t test, P > 5 x 10–3) (Fig. 1). Thus, the VSA expressed by this isolate appeared to be a mixture of VSAPAM and non-PAM VSA (12).
We next measured the ability of IE from the 16 patient isolates to adhere to CSA and to four cell lines purchased from American Type Culture Collection (http://www.lgcpromochem-atcc.com). The lines used were the Chinese hamster ovary (CHO) line K1 (ATCC CCL-61) that expresses CSA, a CHO glycosylation mutant line (A-745; ATCC CRL-2242) that does not express CSA, a CHO cell line transfected with human CD36 (CHO-CD36; ATCC CRL-2092), and the choriocarcinoma cell line BeWo (ATCC CCL-98). All cell lines were maintained as suggested by the American Type Culture Collection. In some preliminary experiments, we pretreated the BeWo cells with forskolin to induce multinucleated syncytia (23). However, forskolin treatment did not markedly affect selection efficiency, and forskolin was not used in the results presented in this paper.
Compared to the CSA-deficient cell line (A-745), the 15 VSAPAM-expressing P. falciparum patient isolates bound significantly better to CSA and to each of the other cell lines (Kruskal-Wallis test, P < 0.001, and Dunn's post hoc test, P < 0.05 in all cases) (Fig. 2). Adhesion of these isolates to CSA and to the CSA-expressing CHO cell lines (CHO-K1 and CHO-CD36) was similar, whereas adhesion to the BeWo line was significantly higher (Kruskal-Wallis test, P < 0.001, and Dunn's post hoc test, P < 0.05 CSA versus BeWo) (Fig. 2). DP168 did not bind well to CSA but bound strongly to CHO-CD36 (Fig. 2). A subline of the long-term in vitro-adapted P. falciparum line FCR3 (FCR3-CHO) that expressed VSAPAM (t test as above, P < 1 x 10–10) following selection for binding to K1 and included as a control for CSA-mediated adhesion (10) bound well to CSA and to all the CSA-expressing lines (Fig. 2). In contrast, a CD36-adhering FCR3 subline (FCR3-CD36) expressing non-PAM VSA (t test as above, P = 0.16) and included as a control line for CD36-mediated binding (10) only bound to the CD36-transfected CHO cell line (Fig. 2). Specifically, FRC3-CD36 did not bind well to the BeWo cell line, which does not express CD36 (20). These results show that VSAPAM-expressing patient isolates bind strongly to BeWo cells, suggesting that this line is a useful tool in selecting for VSAPAM-expression in vitro.
To test this hypothesis, we subjected six long-term in vitro adapted laboratory lines (3D7, Dd2, FCR3, HB-3, K1, and NF54) to selection for adhesion to BeWo cells (Table 1). The lines were chosen because none of them spontaneously expressed VSAPAM and because our attempts to select for VSAPAM expression by 10 rounds of panning on CSA (13) had been unsuccessful for four of them (Table 1). For three of five lines tested, selection for VSAPAM expression by selection for adhesion to the K1 cell line (10) had been equally ineffective (Table 1). After only three rounds of selection for adhesion to BeWo cells, three of the lines had clearly acquired VSAPAM expression (Table 1 and Fig. 3). With respect to 3D7, plasma levels of VSA-specific IgG were lower (paired t test, P = 0.01) in males and higher in multiparous women (P = 0.0001) after three rounds of selection, but substantial male reactivity remained, indicating that acquisition of VSAPAM was incomplete for this clone.
Finally, we subjected three P. falciparum isolates (Busua, N0031, and N0045) recently obtained from nonpregnant malaria patients to selection for IE adhesion to BeWo cells. Two of these isolates rapidly acquired VSAPAM expression (Table 1). Although the VSA expressed by the third isolate clearly changed in response to selection, it did not become VSAPAM-like, and the IE from this isolate appeared to have affinity for the plastic surface of culture plates rather than for receptors on the BeWo cells (data not shown).
In conclusion, we have shown that VSAPAM-expressing P. falciparum patient isolates implicated in the pathogenesis of PAM adhere more strongly to the placental choriocarcinoma cell line BeWo than to CSA and cell lines often used in studies of P. falciparum involved in PAM. Furthermore, we demonstrate that the BeWo cell line is an efficient tool to select for VSAPAM expression in vitro. These findings establish the BeWo cell line as an important tool for research on the VSAPAM that are centrally involved in the pathogenesis of and protective immunity to PAM. Studies on the ability of VSAPAM-specific IgG to interfere with adhesion of VSAPAM-expressing IE are currently under way.
ACKNOWLEDGMENTS
We are indebted to all the individuals who donated parasite and plasma samples for the study. Kirsten Pihl and Maiken Christensen are thanked for excellent technical assistance in the laboratory.
The work received financial support from the Commission of European Union (grant QLK2-CT-2001-01302, PAMVAC), from the Danish Medical Research Council (SSVF; grants 22-02-0571 and 22-03-0333), and from the Danish Research Council for Development Research (RUF; grant 104.Dan.8.L.306). R.H. was supported by a scholarship from SSVF (grant 22-04-0012).
REFERENCES
1. Beeson, J. G., G. V. Brown, M. E. Molyneux, C. Mhango, F. Dzinjalamala, and S. J. Rogerson. 1999. Plasmodium falciparum isolates from infected pregnant women and children are associated with distinct adhesive and antigenic properties. J. Infect. Dis. 180:464-472.
2. Bhasin, V. K., and W. Trager. 1984. Gametocyte-forming and non-gametocyte-forming clones of Plasmodium falciparum. Am. J. Trop. Med. Hyg. 33:534-537.
3. Chaiyaroj, S. C., P. Angkasekwinai, A. Buranakiti, S. Looareesuwan, S. J. Rogerson, and G. V. Brown. 1996. Cytoadherence characteristics of Plasmodium falciparum isolates from Thailand: evidence for chondroitin sulfate A as a cytoadherence receptor. Am. J. Trop. Med. Hyg. 55:76-80.
4. Delemarre, B. J., and H. J. Van der Kaay. 1979. Malaria tropica op natuurlijke wijze verkregen in Nederland. Ned. Tijdschr. Geneeskd. 123:1981-1982.
5. Duffy, P. E., and M. Fried. 2003. Antibodies that inhibit Plasmodium falciparum adhesion to chondroitin sulfate A are associated with increased birth weight and the gestational age of newborns. Infect. Immun. 71:6620-6623.
6. Fried, M., and P. E. Duffy. 1996. Adherence of Plasmodium falciparum to chondroitin sulfate A in the human placenta. Science 272:1502-1504.
7. Hviid, L. 2004. The immuno-epidemiology of pregnancy-associated malaria: a variant surface antigen-specific perspective. Parasite Immunol. 26:477-486.
8. Jensen, J. B., and W. Trager. 1978. Plasmodium falciparum in culture: establishment of additional strains. Am. J. Trop. Med. Hyg. 27:743-746.
9. Lucchi, N. W., R. Koopman, D. S. Peterson, and J. M. Moore. Plasmodium falciparum-infected red blood cells selected for binding to cultured syncytiotrophoblast bind to chondroitin sulfate A and induce tyrosine phosphorylation in the syncytiotrophoblast. Placenta, in press.
10. Megnekou, R., T. Staalsoe, D. W. Taylor, R. Leke, and L. Hviid. 2005. Effects of pregnancy and intensity of Plasmodium falciparum transmission on immunoglobulin G subclass responses to variant surface antigens. Infect. Immun. 73:4112-4118.
11. Oduola, A. M., W. K. Milhous, N. F. Weatherly, J. H. Bowdre, and R. E. Desjardins. 1988. Plasmodium falciparum: induction of resistance to mefloquine in cloned strains by continuous drug exposure in vitro. Exp. Parasitol. 67:354-360.
12. Ofori, M. F., T. Staalsoe, V. Bam, M. Lundquist, K. P. David, E. N. L. Browne, B. D. Akanmori, and L. Hviid. 2003. Expression of variant surface antigens by Plasmodium falciparum parasites in the peripheral blood of clinically immune pregnant women indicates ongoing placental infection. Infect. Immun. 71:1584-1586.
13. Ricke, C. H., T. Staalsoe, K. Koram, B. D. Akanmori, E. M. Riley, T. G. Theander, and L. Hviid. 2000. Plasma antibodies from malaria-exposed pregnant women recognize variant surface antigens on Plasmodium falciparum-infected erythrocytes in a parity-dependent manner and block parasite adhesion to chondroitin sulfate A. J. Immunol. 165:3309-3316.
14. Smith, J. D., and K. W. Deitsch. 2004. Pregnancy-associated malaria and the prospects for syndrome-specific antimalaria vaccines. J. Exp. Med. 200:1093-1097.
15. Staalsoe, T., H. A. Giha, D. Dodoo, T. G. Theander, and L. Hviid. 1999. Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry. Cytometry 35:329-336.
16. Staalsoe, T., R. Megnekou, N. Fievet, C. H. Ricke, H. D. Zornig, R. Leke, D. W. Taylor, P. Deloron, and L. Hviid. 2001. Acquisition and decay of antibodies to pregnancy-associated variant antigens on the surface of Plasmodium falciparum-infected erythrocytes that are associated with protection against placental parasitemia. J. Infect. Dis. 184:618-626.
17. Staalsoe, T., C. E. Shulman, J. N. Bulmer, K. Kawuondo, K. Marsh, and L. Hviid. 2004. Variant surface antigen-specific IgG and protection against the clinical consequences of pregnancy-associated Plasmodium falciparum malaria. Lancet 363:283-289.
18. Steketee, R. W., B. L. Nahlen, M. E. Parise, and C. Menendez. 2001. The burden of malaria in pregnancy in malaria-endemic areas. Am. J. Trop. Med. Hyg. 64:28-35.
19. Thaithong, S., and G. H. Beale. 1981. Resistance of ten Thai isolates of Plasmodium falciparum to chloroquine and pyrimethamine by in vitro tests. Trans. R. Soc. Trop. Med. Hyg. 75:271-273.
20. Viebig, N. K., M. C. Nunes, A. Scherf, and B. Gamain. 2006. The human placental derived BeWo cell line: a useful model for selecting Plasmodium falciparum CSA-binding parasites. Exp. Parasitol. 112:121-125.
21. Walliker, D., I. A. Quakyi, T. E. Wellems, T. F. McCutchan, A. Szarfman, W. T. London, L. M. Corcoran, T. R. Burkot, and R. Carter. 1987. Genetic analysis of the human malaria parasite Plasmodium falciparum. Science 236:1661-1666.
22. Wellems, T. E., L. J. Panton, I. Y. Gluzman, R. Do, V., R. W. Gwadz, A. Walker-Jonah, and D. J. Krogstad. 1990. Chloroquine resistance not linked to mdr-like genes in a Plasmodium falciparum cross. Nature 345:253-255.
23. Wice, B., D. Menton, H. Geuze, and A. L. Schwartz. 1990. Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation in vitro. Exp. Cell Res. 186:306-316.(Rikke N. Haase, Rosette M)