As another piece of evidence that mouse and human EPO/ EPOR signaling are different, Divoky and Prchal point to the production of mice expressing truncated Epor with only a single cytoplasmic tyrosine at 343. 1,2 After initial reports of a negative regulatory domain in the cytoplasmic tail of mouse Epor 11 and human EPOR, 12 other truncated deletions in human EPOR have been associated with polycythemia including the mutation con- structed by Divoky et al. 1 These mice express human EpoR with a truncated deletion just before tyrosine 410 and exhibit a polycythe- mia phenotype as predicted from the human condition. Interest- ingly, in addition to direct alteration of EPO signaling, increased cell-surface expression via posttranslational mechanism(s) has been suggested for a truncated EPOR associated primary familial and congenital polycythemia. 13 Other mice expressing a truncated mouse EpoR were constructed by altering the endogenous Epor sequence 3' of the HindIII site resulting in a truncated mouse Epor with a single cytoplasmic tyrosine that is about 2 dozen amino acids shorter than the Divoky and Prchal deletion. 2 These mice exhibit largely constitutive erythropoiesis and are not polycythe- mic. While it is possible that these differences may be inherent in mouse versus human EPOR, variation in erythrocytosis may also result from the extent or level of expression of the truncations. Recapitulation of the human phenotype with the truncated human EPOR by Divoky et al 1 confirms the similarity of EPO/ EPOR signaling in mouse and human and the potential for using mouse models to understand EPO/EPOR interactions. The differ- ences noted here among the EPOR mouse models illustrate the advantages and limitations of manipulating the mouse genome to alter phenotypic expression. When comparing model systems, careful analysis of gene construction in vivo, mouse strains, and possible integration sites are necessary to ensure comparable expression and analogous gene products. Note that while the original targeting construct for human EPOR by Divoky and Prchal was active in vitro, it was necessary to remove the neomycin (neo)-selectable marker from human EPOR intron 6 to obtain expression in vivo, although the neo gene remains 3' flanking the endogenous mouse Epor produced by Zang et al. 1,2 Variation in gene expression can become significant, particularly when the differences are small. While human and mouse EPO/EPOR signaling may not be equivalent, we do not see any evidence that they are significantly different in our EPO/EPOR animal model. Xiaobing Yu and Constance Tom Noguchi Correspondence: Constance Tom Noguchi, Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 10 Center Dr MSC-1822, Bethesda, MD 20892-1822 References 1. Divoky V, Liu Z, Ryan TM, Prchal JF, Townes TM, Prchal JT. Mouse model of congenital polycythemia: homologous replacement of murine gene by mutant human erythropoietin receptor gene. Proc Natl Acad Sci U S A. 2001;98:986- 991. 2. Zang H, Sato K, Nakajima H, McKay C, Ney PA, Ihle JN. The distal region and receptor tyrosines of the Epo receptor are non-essential for in vivo erythropoi- esis. EMBO J. 2001;20:3156-3166. 3. Liu C, Shen K, Liu Z, Noguchi CT. Regulated human erythropoietin receptor expression in mouse brain. J Biol Chem. 1997;272:32395-32400. 4. Yu X, Lin CS, Costantini F, Noguchi CT. The human erythropoietin receptor gene rescues erythropoiesis and developmental defects in the erythropoietin receptor null mouse. Blood. 2001;98:475-477. 5. Yu X, Shacka JJ, Eells JB, et al. Erythropoietin receptor signalling is required for normal brain development. Development. 2002;129:505-516. 6. Chin K, Oda N, Shen K, Noguchi CT. Regulation of transcription of the human erythropoietin receptor gene by proteins binding to GATA-1 and Sp1 motifs. Nucleic Acids Res. 1995;23:3041-3049. 7. Liu ZY, Chin K, Noguchi CT. Tissue specific expression of human erythropoietin receptor in transgenic mice. Dev Biol. 1994;166:159-169. 8. Zon LI, Youssoufian H, Mather C, Lodish HF, Orkin SH. Activation of the eryth- ropoietin receptor promoter by transcription factor GATA-1. Proc Natl Acad Sci U S A. 1991;88:10638-10641. 9. Noguchi CT, Bae KS, Chin K, Wada Y, Schechter AN, Hankins WD. Cloning of the human erythropoietin receptor gene. Blood. 1991;78:2548-2556. 10. Youssoufian H, Lodish HF. Transcriptional inhibition of the murine erythropoi- etin receptor gene by an upstream repetitive element. Mol Cell Biol. 1993;13: 98-104. 11. D’Andrea AD, Yoshimura A, Youssoufian H, Zon LI, Koo JW, Lodish HF. The cytoplasmic region of the erythropoietin receptor contains nonoverlapping posi- tive and negative growth-regulatory domains. Mol Cell Biol. 1991;11:1980- 1987. 12. de la Chapelle A, Traskelin AL, Juvonen E. Truncated erythropoietin receptor causes dominantly inherited benign human erythrocytosis. Proc Natl Acad Sci U S A. 1993;90:4495-4499. 13. Motohashi T, Nakamura Y, Osawa M, et al. Increased cell surface expression of C-terminal truncated erythropoietin receptors in polycythemia. Eur J Haematol. 2001;67:88-93. To the editor: CD34 stem cell dose and development of extensive chronic graft-versus-host disease Granulocyte colony-stimulating factor (G-CSF)–mobilized periph- eral blood mononuclear cells (G-PBMCs) are increasingly used as a stem cell source for allogeneic transplantation. The in vivo administration of G-CSF to the donor results in an increase of circulating CD34 + hematopoieitic progenitor cells, which are responsible for engraftment after myeloablative therapy. In a recent report by Zaucha et al, 1 an association between the CD34 cell dose in G-PBMCs collected from human leuko- cyte antigen (HLA)–identical siblings and the development of extensive chronic graft-versus-host disease (cGvHD) is shown. The number of CD3 T lymphocytes or CD14 monocytes in the graft were not significantly associated with extensve cGvHD. Therefore, the authors concluded that increasing the CD34 cell number in G-PBMC products may be counterproductive and suggest limiting the number of CD34 cells in the unmodified G-PBMC grafts. Rather than using unmodified G-PBMC grafts, we have trans- plantated highly purified G-CSF–mobilized peripheral CD34 stem cells from HLA-matched unrelated 2 and HLA-mismatched hap- loidentical donors 3 in 76 pediatric patients with malignant and nonmalignant diseases. The CD34 cells were purified to a median purity of at least 98% and transplanted without any posttransplanta- tion GvHD prophylaxis. The median number of transplanted CD34 stem cells was 12.1  10 6 /kg recipient body weight with a wide range of 1 to 54.6  10 6 /kg. The median number of cotransplanted CD3 T lymphocytes was 7.6  10 3 /kg (range, 0.5-130  10 3 /kg). The median follow-up time for our patients is 2.9 years (range, 0.9 years to 5.7 years). Only patients who survived at least 100 days after transplantation were analyzed. In contrast to the study by Zaucha et al, we have not seen any correlation between the number of transplanted CD34 stem cells and the development of extensive chronic GvHD. A transient, CORRESPONDENCE 3875 BLOOD, 15 MAY 2002  VOLUME 99, NUMBER 10 Downloaded from http://ashpublications.org/blood/article-pdf/99/10/3873/1684974/h81002003867e.pdf by guest on 24 July 2022