IDO induction can deplete l-tryptophan in target cells, an effect partially responsible for the antimicrobial activities and antiallogeneic T cell responses of IFN-γ in human macrophages, dendritic cells, and bone marrow cells. l-Tryptophan depletion and NO production are both known to have an antimicrobial effect in macrophages, and the interaction of these two mechanisms is unclear. In this study we found that IDO activity was inhibited by the peroxynitrite generator, 3-(4-morpholinyl)sydnonimine, in PMA-differentiated cytokine-induced THP-1 (acute monocytic leukemia) cells and IFN-γ-stimulated PBMCs, whereas IDO protein expression was unaffected compared with that in untreated cells. Nitrotyrosine was detected in immunoprecipitated (IP)-IDO from PMA-differentiated cytokine-induced THP-1 cells treated with 3-(4-morpholinyl)sydnonimine, but not from untreated cells. Treatment of IP-IDO and recombinant IDO (rIDO) with peroxynitrite significantly decreased enzyme activity. Nitrotyrosine was detected in both peroxynitrite-treated IP-IDO and rIDO, but not in either untreated IP-IDO or rIDO. Peptide analysis by liquid chromatography/electrospray ionization and tandem mass spectrometry demonstrated that Tyr15, Tyr345, and Tyr353 in rIDO were nitrated by peroxynitrite. The levels of Tyr nitration and the inhibitory effect of peroxynitrite on IDO activity were significantly reduced in the Tyr15-to-Phe mutant. These results indicate that IDO is nitrated and inactivated by peroxynitrite and that nitration of Tyr15 in IDO protein is the most important factor in the inactivation of IDO.

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Objective-Sickle cell anemia is a genetic blood disease resulting from production of mutant β-globin (β S ) and has severe clinical consequences. It is known that a higher cellular γ-globin level, e.g., higher ratio of cellular γ-globin to β S -globin (γ/β S ratio), inhibits sickle hemoglobin (HbS) polymerization tendency. Hence, therapeutic treatment of sickle cell anemia has been focused on introducing γ-globin gene into red blood cells to increase the cellular γ/β S ratio. Here, we have introduced ribozymes and small interfering RNAs (siRNAs) against β S -globin mRNA into blood cells as a means to increase the γ/β S ratio.Methods-Single and multi-ribozymes against β S -globin mRNA have been tested in vitro and in human erythroleukemia K562β S cells that stably express exogenous β S -globin gene. Primary human hematopoietic progenitor cells were also transfected with multi-ribozyme and the γ/(γ+β) ratio determined and compared with cells transfected with long hairpin β-globin cDNA and synthetic siRNA genes.Results-We have found that the multi-ribozyme zb21A containing two ribozyme units effectively reduces β S -globin mRNA both in vitro and in K562β S cells. The γ-globin mRNA to β S -globin mRNA ratio in the multi-ribozyme transfected cells is about a factor of 2 more than that in the control cells. We have also found that the γ/(γ+β) ratio in the transfected hematopoietic progenitor cells is increased by more than 2-fold in cells treated with multi-ribozyme zb21A or siRNA ib5.Conclusion-Our results suggest that introducing multi-ribozymes or siRNAs into red blood cells are comparable in their effectiveness to increase the ratio of cellular γ-globin mRNA to β-or β Sglobin mRNA, providing possible strategies to increase the effectiveness of γ-globin gene transfer as gene therapy for treatment of patients with sickle cell anemia.Sickle cell anemia is an inheritable blood disease caused by a single nucleotide mutation in the human β-globin gene (codon 6 GAG→GTG). Individuals homozygous for this mutant β-globin gene, designated as β S -globin, have severe clinical symptoms, because the abnormal hemoglobin, sickle cell hemoglobin [HbS (β6 Glu→Val)], produced by this gene can polymerize under low oxygen tension, change red blood cell rheology and shape (sickle), and result in vasoocclusive crisis, infarction, and organ damage [1]. It is known that in some patients with milder sickle cell anemia symptoms, there are higher fetal hemoglobin [HbF (α 2 γ 2 )] levels Offprint request to: Dr. Chien Ho, Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh PA 15213; E-mail address: chienho@andrew.cmu.edu.. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process...

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