DNA Topoisomerase II b and Neural Development

Garnham, Malaria Parasites and Other Haemosporidia (Blackwell Scientific, Oxford, 1966)]. Sex ratios based on counts of 50 to 75 gametocytes were found to be representative. We calculated sex ratios from either 50,000 RBCs or 100 gametocytes, whichever was less. Sex ratios are given as proportion of males. 7. R. E. Sinden, G. A. Butcher, O. Billker, S. L. Fleck,. Adv. Parasitol. 38, 53 (1996). 8. R. Carter, R. W. Gwadz, F. M. McAuliffe, Exp. Parasitol. 47, 185 (1979). 9. Infection classification by day with respect to peak parasitemia is retrospective, where P 2 1, P 2 2, and P 2 3 are 1, 2, and 3 days before peak parasitemia. Erythropoietic treatments were carried out on the first day parasites appeared in the blood (parasite patency) and infection peak was not always the same number of days after parasite patency. Hence, treatment with respect to day of peak parasitemia varied, and that information is supplied in the figure legends. Such variation was taken into account in the statistical analyses. 10. Polynomial regression was performed in SIMSTAT for Windows (Provalis Research). 11. K. P. Day, R. E. Hayward, M. Dyer, Parasitology 116, S95 (1998). 12. Two vaccination procedures were carried out according to protocols previously established (8) [R. Carter, R. W. Gwadz, I. Green, Exp. Parasitol. 47, 194 (1979); D. C. Kaushal et al., J. Immunol. 131, 2557 (1983)]. Chickens received intravenous injections with x-irradiated (35 Krad) purified male gametes or a mixture of female gametes and microgametocytes, which are not separable. Each vaccination type was administered three times over 3 weeks per chicken host (n 5 3 for both treatments) before intravenous injection of live parasitized blood. 13. Statistical analyses were conducted with the statistical package Genstat 5.4.1. Because each individual vertebrate host was included in the data set many times, we corrected for repeated measures by fitting a generalized linear mixed model (GLMM procedure) with a Poisson error structure with “animal” as the only term in the random model. For both the sex ratio and oocyst analyses, the data were overdispersed and so were corrected for by estimating a dispersion parameter for each analysis [M. J. Crawley, GLIM for Ecologists (Blackwell Scientific, Oxford, 1993)]. All analyses of the effects of sex ratio on infectivity were controlled for gametocyte density, infection outcome (live or die), day of infection with respect to day of peak parasitemia, and individual host. Statistical significance was presented as Walds statistics, which are equivalent to a x2 analysis. Between-treatment comparisons were performed with respect to peak parasitemia up to, but not including, the day of peak parasitemia, at which time any erythropoietic treatment effects were disguised by the erythropoietic response normally associated with infection control. For clarity, Figs. 1 and 4 show treatment means. 14. Fiveto seven-day-old Aedes aegypti (Liverpool Blackeye strain) were used for the mosquito infection studies. Oocyst counts in mosquitoes were made 7 days postinfection on midguts dissected from 30 gravid females and then stained with 0.5% mercurochrome. 15. Mosquito infectivity: between-treatment comparisons were done by day with respect to peak parasitemia specifying a Poisson error structure (which gives the same fit as a negative binomial) [K. Wilson, B. T. Grenfell, D. J. Shaw, Funct. Ecol. 10, 592 (1996)] for the distribution of oocyst counts per mosquito per chicken. For clarity, Fig. 3 shows treatment geometric means with 99% confidence intervals. 16. F. Hawking, K. Gammage, M. J. Worms, Trans. R. Soc. Trop. Med. Hyg. 65, 189 (1972). 17. Reticulocyte-rich blood was injected intravenously in exchange for blood (20% of total blood volume) taken from an infected chicken host, thereby increasing the proportion of RBCs that were reticulocytes without stimulating erythropoiesis (n 5 6). Reticulocytes were obtained from chickens 72 hours after subcutaneous injection of the hemolytic drug phenylhydrazine hydrochloride (Sigma) at a dose of 6 mg per 100 g of body weight; the blood was washed in 13 phosphate-buffered saline (0.14 M NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8,1 mM Na2 HPO4), and the volume was restored with serum from naive chickens. 18. S. L. Schindler and R. P. Gildersleeve, Comp. Biochem. Physiol. A Comp. Physiol. 87, 533 (1987). 19. Two methods were used to induce erythropoiesis: exsanguination of 20% to 30% of total blood volume by cardiac puncture on the day of parasite patency (.0.1% parasitemia) (n 5 6), and 16 hours of exposure to mildly hypoxic conditions on the day of patency (15% oxygen, 85% nitrogen; Carboxyque, France) (n 5 8); W. F. Rosse and T. A. Waldmann, Blood 27, 654 (1966). 20. J. S. Milledge and P. M. Cotes, J. Appl. Physiol. 59, 360 (1985); H. Pagel, A. Engel, W. Jelkmann, Adv. Exp. Med. Biol. 317, 515 (1992); M. J. McGuire and J. L. Spivak, in The Fundamentals of Clinical Hematology, J. L. Spivak and E. R. Eichner, Eds. ( Johns Hopkins Univ. Press, London, 1993), pp. 117–128. 21. R. E. L. Paul, A. Raibaud, P. T. Brey, Parassitologia 41, 153 (1999). 22. W. Jelkmann, Clin. Invest. 72, S3 (1994). 23. Two methods were used to induce erythropoiesis in mice: intraperitoneal injection of mouse recombinant Epo (Boehringer Mannheim, France) at a concentration of 70 or 700 units/liter administered in 100 ml of 0.9% NaCl on the 3 days after parasite patency, and 16 hours of exposure to mildly hypoxic conditions on the day of patency (15% oxygen, 85% nitrogen) (n 5 5 for each treatment and n 5 10 for control infections). 24. E. Geiringer, Proc. Soc. Exp. Biol. Med. 106, 752 (1961). 25. A. M. el Hassan, A. M. Saeed, J. Fandrey, W. Jelkmann, Eur. J. Haematol. 59, 299 (1997). 26. Supported by Institut Pasteur, Paris. R.E.L.P. was supported by the Medical Research Council (UK)/CNRS, Fondation pour la Recherche Médicale, and a European Commission Training and Mobility of Researchers Marie Curie Research Training Grant. We thank A. Danchin, G. Milon, P. Langlade-Demoyen, C. Roth, P. Druilhe, C. Lazure, K. Kean, and S. Cole for helpful discussions and A. Carmi-Leroy for mosquito rearing.