Research Article
Abstract
We treated 10 children with X-linked SCID (SCID-X1) using gammaretrovirus-mediated gene transfer. Those with sufficient follow-up were found to have recovered substantial immunity in the absence of any serious adverse events up to 5 years after treatment. To determine the influence of vector integration on lymphoid reconstitution, we compared retroviral integration sites (RISs) from peripheral blood CD3+ T lymphocytes of 5 patients taken between 9 and 30 months after transplantation with transduced CD34+ progenitor cells derived from 1 further patient and 1 healthy donor. Integration occurred preferentially in gene regions on either side of transcription start sites, was clustered, and correlated with the expression level in CD34+ progenitors during transduction. In contrast to those in CD34+ cells, RISs recovered from engrafted CD3+ T cells were significantly overrepresented within or near genes encoding proteins with kinase or transferase activity or involved in phosphorus metabolism. Although gross patterns of gene expression were unchanged in transduced cells, the divergence of RIS target frequency between transduced progenitor cells and post-thymic T lymphocytes indicates that vector integration influences cell survival, engraftment, or proliferation.
Authors
Kerstin Schwarzwaelder, Steven J. Howe, Manfred Schmidt, Martijn H. Brugman, Annette Deichmann, Hanno Glimm, Sonja Schmidt, Claudia Prinz, Manuela Wissler, Douglas J.S. King, Fang Zhang, Kathryn L. Parsley, Kimberly C. Gilmour, Joanna Sinclair, Jinhua Bayford, Rachel Peraj, Karin Pike-Overzet, Frank J.T. Staal, Dick de Ridder, Christine Kinnon, Ulrich Abel, Gerard Wagemaker, H. Bobby Gaspar, Adrian J. Thrasher, Christof von Kalle
Abstract
Gene transfer into HSCs is an effective treatment for SCID, although potentially limited by the risk of insertional mutagenesis. We performed a genome-wide analysis of retroviral vector integrations in genetically corrected HSCs and their multilineage progeny before and up to 47 months after transplantation into 5 patients with adenosine deaminase–deficient SCID. Gene-dense regions, promoters, and transcriptionally active genes were preferred retroviral integrations sites (RISs) both in preinfusion transduced CD34+ cells and in vivo after gene therapy. The occurrence of insertion sites proximal to protooncogenes or genes controlling cell growth and self renewal, including LMO2, was not associated with clonal selection or expansion in vivo. Clonal analysis of long-term repopulating cell progeny in vivo revealed highly polyclonal T cell populations and shared RISs among multiple lineages, demonstrating the engraftment of multipotent HSCs. These data have important implications for the biology of retroviral vectors, the dynamics of genetically modified HSCs, and the safety of gene therapy.
Authors
Alessandro Aiuti, Barbara Cassani, Grazia Andolfi, Massimiliano Mirolo, Luca Biasco, Alessandra Recchia, Fabrizia Urbinati, Cristina Valacca, Samantha Scaramuzza, Memet Aker, Shimon Slavin, Matteo Cazzola, Daniela Sartori, Alessandro Ambrosi, Clelia Di Serio, Maria Grazia Roncarolo, Fulvio Mavilio, Claudio Bordignon
Abstract
Primary hypomagnesemia constitutes a rare heterogeneous group of disorders characterized by renal or intestinal magnesium (Mg2+) wasting resulting in generally shared symptoms of Mg2+ depletion, such as tetany and generalized convulsions, and often including associated disturbances in calcium excretion. However, most of the genes involved in the physiology of Mg2+ handling are unknown. Through the discovery of a mutation in the EGF gene in isolated autosomal recessive renal hypomagnesemia, we have, for what we believe is the first time, identified a magnesiotropic hormone crucial for total body Mg2+ balance. The mutation leads to impaired basolateral sorting of pro-EGF. As a consequence, the renal EGFR is inadequately stimulated, resulting in insufficient activation of the epithelial Mg2+ channel TRPM6 (transient receptor potential cation channel, subfamily M, member 6) and thereby Mg2+ loss. Furthermore, we show that colorectal cancer patients treated with cetuximab, an antagonist of the EGFR, develop hypomagnesemia, emphasizing the significance of EGF in maintaining Mg2+ balance.
Authors
Wouter M. Tiel Groenestege, Stéphanie Thébault, Jenny van der Wijst, Dennis van den Berg, Rob Janssen, Sabine Tejpar, Lambertus P. van den Heuvel, Eric van Cutsem, Joost G. Hoenderop, Nine V. Knoers, René J. Bindels
Abstract
The initiation of mammalian puberty and the maintenance of female reproductive cycles are events controlled by hypothalamic neurons that secrete the decapeptide gonadotropin-releasing hormone (GnRH). GnRH secretion is, in turn, controlled by changes in neuronal and glial inputs to GnRH-producing neurons. The hierarchical control of the process is unknown, but it requires coordinated regulation of these cell-cell interactions. Here we report the functional characterization of a gene (termed enhanced at puberty 1 [EAP1]) that appears to act as an upstream transcriptional regulator of neuronal networks controlling female reproductive function. EAP1 expression increased selectively at puberty in both the nonhuman primate and rodent hypothalamus. EAP1 encoded a nuclear protein expressed in neurons involved in the inhibitory and facilitatory control of reproduction. EAP1 transactivated genes required for reproductive function, such as GNRH1, and repressed inhibitory genes, such as preproenkephalin. It contained a RING finger domain of the C3HC4 subclass required for this dual transcriptional activity. Inhibition of EAP1 expression, targeted to the rodent hypothalamus via lentivirus-mediated delivery of EAP1 siRNAs, delayed puberty, disrupted estrous cyclicity, and resulted in ovarian abnormalities. These results suggest that EAP1 is a transcriptional regulator that, acting within the neuroendocrine brain, contributes to controlling female reproductive function.
Authors
Sabine Heger, Claudio Mastronardi, Gregory A. Dissen, Alejandro Lomniczi, Ricardo Cabrera, Christian L. Roth, Heike Jung, Francesco Galimi, Wolfgang Sippell, Sergio R. Ojeda
Abstract
Erythroid cells accumulate hemoglobin as they mature and as a result are highly prone to oxidative damage. However, mechanisms of transcriptional control of antioxidant defense in erythroid cells have thus far been poorly characterized. We observed that animals deficient in the forkhead box O3 (Foxo3) transcription factor died rapidly when exposed to erythroid oxidative stress–induced conditions, while wild-type mice showed no decreased viability. In view of this striking finding, we investigated the potential role of Foxo3 in the regulation of ROS in erythropoiesis. Foxo3 expression, nuclear localization, and transcriptional activity were all enhanced during normal erythroid cell maturation. Foxo3-deficient erythrocytes exhibited decreased expression of ROS scavenging enzymes and had a ROS-mediated shortened lifespan and evidence of oxidative damage. Furthermore, loss of Foxo3 induced mitotic arrest in erythroid precursor cells, leading to a significant decrease in the rate of in vivo erythroid maturation. We identified ROS-mediated upregulation of p21CIP1/WAF1/Sdi1 (also known as Cdkn1a) as a major contributor to the interference with cell cycle progression in Foxo3-deficient erythroid precursor cells. These findings establish an essential nonredundant function for Foxo3 in the regulation of oxidative stress, cell cycle, maturation, and lifespan of erythroid cells. These results may have an impact on the understanding of human disorders in which ROS play a role.
Authors
Dragan Marinkovic, Xin Zhang, Safak Yalcin, Julia P. Luciano, Carlo Brugnara, Tara Huber, Saghi Ghaffari
Abstract
Liver X receptors (LXRs) α and β are transcriptional regulators of cholesterol homeostasis and potential targets for the development of antiatherosclerosis drugs. However, the specific roles of individual LXR isotypes in atherosclerosis and the pharmacological effects of synthetic agonists remain unclear. Previous work has shown that mice lacking LXRα accumulate cholesterol in the liver but not in peripheral tissues. In striking contrast, we demonstrate here that LXRα–/–apoE–/– mice exhibit extreme cholesterol accumulation in peripheral tissues, a dramatic increase in whole-body cholesterol burden, and accelerated atherosclerosis. The phenotype of these mice suggests that the level of LXR pathway activation in macrophages achieved by LXRβ and endogenous ligand is unable to maintain homeostasis in the setting of hypercholesterolemia. Surprisingly, however, a highly efficacious synthetic agonist was able to compensate for the loss of LXRα. Treatment of LXRα–/–apoE–/– mice with synthetic LXR ligand ameliorates the cholesterol overload phenotype and reduces atherosclerosis. These observations indicate that LXRα has an essential role in maintaining peripheral cholesterol homeostasis in the context of hypercholesterolemia and provide in vivo support for drug development strategies targeting LXRβ.
Authors
Michelle N. Bradley, Cynthia Hong, Mingyi Chen, Sean B. Joseph, Damien C. Wilpitz, Xuping Wang, Aldons J. Lusis, Allan Collins, Willa A. Hseuh, Jon L. Collins, Rajendra K. Tangirala, Peter Tontonoz
Abstract
The inhibition of apoptosis of infected host cells is a well-known but poorly understood function of pathogenic mycobacteria. We show that inactivation of the secA2 gene in Mycobacterium tuberculosis, which encodes a component of a virulence-associated protein secretion system, enhanced the apoptosis of infected macrophages by diminishing secretion of mycobacterial superoxide dismutase. Deletion of secA2 markedly increased priming of antigen-specific CD8+ T cells in vivo, and vaccination of mice and guinea pigs with a secA2 mutant significantly increased resistance to M. tuberculosis challenge compared with standard M. bovis bacille Calmette-Guérin vaccination. Our results define a mechanism for a key immune evasion strategy of M. tuberculosis and provide what we believe to be a novel approach for improving mycobacterial vaccines.
Authors
Joseph Hinchey, Sunhee Lee, Bo Y. Jeon, Randall J. Basaraba, Manjunatha M. Venkataswamy, Bing Chen, John Chan, Miriam Braunstein, Ian M. Orme, Steven C. Derrick, Sheldon L. Morris, William R. Jacobs Jr., Steven A. Porcelli
Abstract
Injection of apoptotic cells can induce suppression of immune responses to cell-associated antigens. Here, we show that intravenous injection of apoptotic cells expressing a fragment of myelin oligodendrocyte glycoprotein (MOG) reduced MOG-specific T cell response and prevented the development of EAE. Since injected apoptotic cells accumulated initially in the splenic marginal zone (MZ), the role of macrophages in the MZ in immune suppression was examined using transgenic mice in which these cells could be transiently deleted by diphtheria toxin (DT) injection. DT-treated mice became susceptible to EAE even though MOG-expressing apoptotic cells were preinjected. Deletion of the macrophages caused delayed clearance of injected dying cells in the MZ. In wild-type mice, injected apoptotic cells were selectively engulfed by CD8α+ DCs, which are responsible for suppression of immune responses to cell-associated antigens. In contrast, deletion of macrophages in the MZ caused aberrant phagocytosis of injected dying cells by CD8α–CD11b+ DCs. These results indicate that macrophages in the MZ regulate not only efficient clearance of apoptotic cells but also selective engulfment of dying cells by CD8α+ DCs and that functional failure of these unique macrophages impairs suppression of immune responses to cell-associated antigens.
Authors
Yasunobu Miyake, Kenichi Asano, Hitomi Kaise, Miho Uemura, Manabu Nakayama, Masato Tanaka
Abstract
Kidney podocytes and their foot processes maintain the ultrafiltration barrier and prevent urinary protein loss (proteinuria). Here we show that the GTPase dynamin is essential for podocyte function. During proteinuric kidney disease, induction of cytoplasmic cathepsin L leads to cleavage of dynamin at an evolutionary conserved site, resulting in reorganization of the podocyte actin cytoskeleton and proteinuria. Dynamin mutants that lack the cathepsin L site, or render the cathepsin L site inaccessible through dynamin self-assembly, are resistant to cathepsin L cleavage. When delivered into mice, these mutants restored podocyte function and resolve proteinuria. Our study identifies dynamin as a critical regulator of renal permselectivity that is specifically targeted by proteolysis under pathological conditions.
Authors
Sanja Sever, Mehmet M. Altintas, Sharif R. Nankoe, Clemens C. Möller, David Ko, Changli Wei, Joel Henderson, Elizabetta C. del Re, Lianne Hsing, Ann Erickson, Clemens D. Cohen, Matthias Kretzler, Dontscho Kerjaschki, Alexander Rudensky, Boris Nikolic, Jochen Reiser
Abstract
Acute spinal cord injury (SCI) causes progressive hemorrhagic necrosis (PHN), a poorly understood pathological process characterized by hemorrhage and necrosis that leads to devastating loss of spinal cord tissue, cystic cavitation of the cord, and debilitating neurological dysfunction. Using a rodent model of severe cervical SCI, we tested the hypothesis that sulfonylurea receptor 1–regulated (SUR1-regulated) Ca2+-activated, [ATP]i-sensitive nonspecific cation (NCCa-ATP) channels are involved in PHN. In control rats, SCI caused a progressively expansive lesion with fragmentation of capillaries, hemorrhage that doubled in volume over 12 hours, tissue necrosis, and severe neurological dysfunction. SUR1 expression was upregulated in capillaries and neurons surrounding necrotic lesions. Patch clamp of cultured endothelial cells exposed to hypoxia showed that upregulation of SUR1 was associated with expression of functional SUR1-regulated NCCa-ATP channels. Following SCI, block of SUR1 by glibenclamide or repaglinide or suppression of Abcc8, which encodes for SUR1 by phosphorothioated antisense oligodeoxynucleotide essentially eliminated capillary fragmentation and progressive accumulation of blood, was associated with significant sparing of white matter tracts and a 3-fold reduction in lesion volume, and resulted in marked neurobehavioral functional improvement compared with controls. We conclude that SUR1-regulated NCCa-ATP channels in capillary endothelium are critical to development of PHN and constitute a major target for therapy in SCI.
Authors
J. Marc Simard, Orest Tsymbalyuk, Alexander Ivanov, Svetlana Ivanova, Sergei Bhatta, Zhihua Geng, S. Kyoon Woo, Volodymyr Gerzanich
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