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TALEN-mediated functional correction of X-linked chronic granulomatous disease in patient-derived induced pluripotent stem cells.
Dreyer, AK, Hoffmann, D, Lachmann, N, Ackermann, M, Steinemann, D, Timm, B, Siler, U, Reichenbach, J, Grez, M, Moritz, T, et al
Biomaterials. 2015;:191-200
Abstract
X-linked chronic granulomatous disease (X-CGD) is an inherited disorder of the immune system. It is characterized by a defect in the production of reactive oxygen species (ROS) in phagocytic cells due to mutations in the NOX2 locus, which encodes gp91phox. Because the success of retroviral gene therapy for X-CGD has been hampered by insertional activation of proto-oncogenes, targeting the insertion of a gp91phox transgene into potential safe harbor sites, such as AAVS1, may represent a valid alternative. To conceptually evaluate this strategy, we generated X-CGD patient-derived induced pluripotent stem cells (iPSCs), which recapitulate the cellular disease phenotype upon granulocytic differentiation. We examined AAVS1-specific zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for their efficacy to target the insertion of a myelo-specific gp91phox cassette to AAVS1. Probably due to their lower cytotoxicity, TALENs were more efficient than ZFNs in generating correctly targeted iPSC colonies, but all corrected iPSC clones showed no signs of mutations at the top-ten predicted off-target sites of both nucleases. Upon differentiation of the corrected X-CGD iPSCs, gp91phox mRNA levels were highly up-regulated and the derived granulocytes exhibited restored ROS production that induced neutrophil extracellular trap (NET) formation. In conclusion, we demonstrate that TALEN-mediated integration of a myelo-specific gp91phox transgene into AAVS1 of patient-derived iPSCs represents a safe and efficient way to generate autologous, functionally corrected granulocytes.
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2.
The effects of gp100 and tyrosinase peptide vaccinations on nevi in melanoma patients.
Cassarino, DS, Miller, WJ, Auerbach, A, Yang, A, Sherry, R, Duray, PH
Journal of cutaneous pathology. 2006;(5):335-42
Abstract
BACKGROUND A new approach to prevent disease recurrence in high-risk melanoma patients involves immunization with gp100 and tyrosinase peptides. This is the first study to examine the effects of such treatments on nevi. DESIGN We studied biopsies of 'clinically atypical' nevi from 10 patients before and after peptide vaccination. All had a cutaneous melanoma measuring at least 1.5 mm in depth, satellite metastases, or at least one positive lymph node. We performed immunohistochemical stains for CD3, CD4, CD8, MHC-I, MHC-II, CD1a, HMB-45, MART-1, tyrosinase, bcl-2, p53, and Ki-67 (mib-1). RESULTS Immunohistochemistry showed no differences in staining due to vaccination in either the immunologic or melanocytic markers. However, there was a significant increase in both p53 and bcl-2 staining, and a trend toward decreased Ki-67 staining, in the nevi post-treatment. DISCUSSION The primary goal of peptide vaccinations with gp100 and tyrosinase is to activate melanoma-specific T cells in order to prevent melanoma recurrence. Nevi were studied in order to assess the effects on benign melanocytes. No significant changes in lymphocytes, langerhans cells, expression of MHC antigens, or melanocytic markers were found. The increase in p53 and bcl-2 raises the possibility that vaccination with melanocytic antigens stimulates a response in benign melanocytes.
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A phase I trial of humanized monoclonal antibody A33 in patients with colorectal carcinoma: biodistribution, pharmacokinetics, and quantitative tumor uptake.
Scott, AM, Lee, FT, Jones, R, Hopkins, W, MacGregor, D, Cebon, JS, Hannah, A, Chong, G, U, P, Papenfuss, A, et al
Clinical cancer research : an official journal of the American Association for Cancer Research. 2005;(13):4810-7
Abstract
PURPOSE To determine the in vivo characteristics of huA33, a CDR-grafted humanized antibody against the A33 antigen, we have conducted an open-label, dose escalation, biopsy-based phase I trial of huA33 in patients with colorectal carcinoma. EXPERIMENTAL DESIGN Patients with colorectal carcinoma were infused with [131I]huA33 (400 MBq: 10 mCi) and [125I]huA33 (40 MBq: 1 mCi) 1 week before surgery. There were four huA33 dose levels (0.25, 1.0, 5.0, and 10 mg/m2). Adverse events, pharmacokinetics, biodistribution, tumor biopsies, and immune responses to huA33 were evaluated. RESULTS There were 12 patients entered into the trial (6 males and 6 females; age range, 39-66 years). No dose-limiting toxicity was observed. The biodistribution of huA33 showed excellent uptake of [131I]huA33 in metastatic colorectal carcinoma. Pharmacokinetic analysis showed no significant difference in terminal half-life (T1/2beta) between dose levels (mean +/- SD, 86.92 +/- 22.12 hours). Modeling of colon uptake of huA33 showed a T1/2 of elimination of 32.4 +/- 8.1 hours. Quantitative tumor uptake ranged from 2.1 x 10(-3) to 11.1 x 10(-3) %ID/g, and tumor/normal tissue and tumor/serum ratios reached as high as 16.3:1 and 4.5:1, respectively. Biosensor analysis detected low-level human anti-human antibody responses in four patients following huA33 infusion. CONCLUSIONS huA33 shows selective and rapid localization to colorectal carcinoma in vivo and penetrates to the center of large necrotic tumors, and colon elimination half-life of huA33 is equivalent to basal colonocyte turnover. The excellent targeting characteristics of this humanized antibody indicate potential for the targeted therapy of metastatic colorectal cancer in future trials.
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Alterations in Fas (CD 95/Apo-1) and Fas ligand (CD178) expression in acute promyelocytic leukemia during treatment with ATRA.
Salih, HR, Kiener, PA
Leukemia & lymphoma. 2004;(1):55-9
Abstract
Over the recent years, treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) has become a widely accepted therapeutic regimen and is considered a model of differentiation therapy in malignant diseases. However, the role of ATRA treatment beyond that of the induction of differentiation of leukemic blasts is still far from being fully understood. Data from in vivo and in vitro studies have demonstrated that during ATRA treatment of APL there are significant changes not only in the expression of the apoptotic molecules Fas and Fas ligand, but also in the expression of other molecules involved both in the regulation of apoptosis and in interactions between host immune and leukemia cells. These effects may thus contribute, at least in part, to the beneficial effects of ATRA therapy in APL. In this report we review the current status of studies that contribute to our understanding of treatment with ATRA. We focus on ATRA-induced changes in apoptotic pathways, particularly as it relates to the Fas/Fas ligand system.
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5.
Variants of toll-like receptor 4 modify the efficacy of statin therapy and the risk of cardiovascular events.
Boekholdt, SM, Agema, WR, Peters, RJ, Zwinderman, AH, van der Wall, EE, Reitsma, PH, Kastelein, JJ, Jukema, JW, ,
Circulation. 2003;(19):2416-21
Abstract
BACKGROUND Atherosclerosis is increasingly considered to be a chronic inflammatory process. We examined whether genetic variants of the toll-like receptor 4 (TLR4), which are correlated with impaired innate immunity and with progression of carotid atherosclerosis, are also associated with coronary atherosclerosis and predict the risk of cardiovascular events. METHODS AND RESULTS Two polymorphisms of the TLR4 gene (Asp299Gly and Thr399Ile) were determined in 655 men with angiographically documented coronary atherosclerosis. All patients participated in a prospective cholesterol-lowering trial evaluating the effect on coronary artery disease and were randomly assigned to either pravastatin or placebo for 2 years. There were no significant differences between genetically defined subgroups with respect to baseline risk factors, treatment, or in-trial changes of lipid, lipoprotein, or angiographic measurements. Genotype was not associated with progression of atherosclerosis. In the pravastatin group, 299Gly carriers had a lower risk of cardiovascular events during follow-up than noncarriers (2.0% versus 11.5%, P=0.045). Among noncarriers, pravastatin reduced the risk of cardiovascular events from 18.1% to 11.5% (P=0.03), whereas among 299Gly carriers this risk was strikingly reduced from 29.6% to 2.0% (P=0.0002, P=0.025 for interaction). CONCLUSIONS Among symptomatic men with documented coronary artery disease, the TLR4 Asp299Gly polymorphism was associated with the risk of cardiovascular events. This variant also modified the efficacy of pravastatin in preventing cardiovascular events, such that carriers of the variant allele had significantly more benefit from pravastatin treatment.
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6.
Chemical neuroanatomy of the vesicular amine transporters.
Weihe, E, Eiden, LE
FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2000;(15):2435-49
Abstract
Acetylcholine, catecholamines, serotonin, and histamine are classical neurotransmitters. These small molecules also play important roles in the endocrine and immune/inflammatory systems. Serotonin secreted from enterochromaffin cells of the gut epithelium regulates gut motility; histamine secreted from basophils and mast cells is a major regulator of vascular permeability and skin inflammatory responses; epinephrine is a classical hormone released from the adrenal medulla. Each of these molecules is released from neural, endocrine, or immune/inflammatory cells only in response to specific physiological stimuli. Regulated secretion is possible because amines are stored in secretory vesicles and released via a stimulus-dependent exocytotic event. Amine storage-at concentrations orders of magnitude higher than in the cytoplasm-is accomplished in turn by specific secretory vesicle transporters that recognize the amines and move them from the cytosol into the vesicle. Immunohistochemical visualization of specific vesicular amine transporters (VATs) in neuronal, endocrine, and inflammatory cells provides important new information about how amine-handling cell phenotypes arise during development and how vesicular transport is regulated during homeostatic response events. Comparison of the chemical neuroanatomy of VATs and amine biosynthetic enzymes has also revealed cell groups that express vesicular transporters but not enzymes for monoamine synthesis, and vice versa: their function and regulation is a new topic of investigation in mammalian neurobiology. The chemical neuroanatomy of the vesicular amine transporters is reviewed here. These and similar data emerging from the study of the localization of the recently characterized vesicular inhibitory and excitatory amino acid transporters will contribute to understanding chemically coded synaptic circuitry in the brain, and amine-handling neuroendocrine and immune/inflammatory cell regulation.