Early studies used serum from rabbits immunized with concentrated EPO-containing urine to achieve neutralization of endogenous EPO in recipient rabbits (25-27). Passively immunized rabbits and mice developed anemia. In a more recent study involving active rather than passive immunization, monkeys treated with a human (Hu)GM-CSF-EPO fusion moiety developed anti-EPO (but not anti-GM-CSF) antibodies (Ab), with resultant anemia (28) (Table 2).

Mice with targeted disruption of the EPO gene or EPO receptor (EPOR) gene develop similar phenotypes. EPO-/- and EPOR-/- embryos die in utero at d 13.5 with failure of fetal liver erythropoiesis (29) and with cardiac defects including ventricular hypoplasia and epicardial and vascular abnormalities (30). Although the EPO-/- and EPOR-/- mice had erythropoietic failure, fetal liver erythroid blast-forming units (BFU-E) and erythroid-colony-forming units (CFU-E) progenitor cells were isolated and capable of terminal differentiation in vitro, implicating EPO in the terminal proliferation and survival of erythroid lineage cells (29). Comprehensive analysis of EPOR+/- mice showed evidence of haploinsufficiency, with lower hematocrits and reduced CFU-E frequencies in both bone marrow and spleen (31).

A human EPO mutant in which Arg103 is replaced by Ala [Epo(R103A)] acts as a competitive inhibitor of EPO in vitro in human EPO signaling systems; its effects in vivo and in murine systems have not been reported, although an intent to study the molecule in animal models was foreshadowed (32).

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