Publications by authors named "Jonathan Werner"

30 Publications

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Scientific and Regulatory Policy Committee Best Practices: Documentation of Sexual Maturity by Microscopic Evaluation in Nonclinical Safety Studies.

Toxicol Pathol 2021 Mar 4:192623321990631. Epub 2021 Mar 4.

510456Idorsia Pharmaceuticals Limited, Allschwil, Switzerland.

The sexual maturity status of animals in nonclinical safety studies can have a significant impact on the microscopic assessment of the reproductive system, the interpretation of potential test article-related findings, and ultimately the assessment of potential risk to humans. However, the assessment and documentation of sexual maturity for animals in nonclinical safety studies is not conducted in a consistent manner across the pharmaceutical and chemical industries. The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology convened an international working group of pathologists and nonclinical safety scientists with expertise in the reproductive system, pathology nomenclature, and Standard for Exchange of Nonclinical Data requirements. This article describes the best practices for documentation of the light microscopic assessment of sexual maturity in males and females for both rodent and nonrodent nonclinical safety studies. In addition, a review of the microscopic features of the immature, peripubertal, and mature male and female reproductive system and general considerations for study types and reporting are provided to aid the study pathologist tasked with documentation of sexual maturity.
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http://dx.doi.org/10.1177/0192623321990631DOI Listing
March 2021

Publication Categories in .

Toxicol Pathol 2021 Feb 12:192623321992305. Epub 2021 Feb 12.

Comparative and Molecular Pathogenesis Branch, National Toxicology Program, 6857National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA.

is the official journal of the Society of Toxicologic Pathology (STP), the British Society of Toxicological Pathology, and the European STP (ESTP). publishes articles related to topics in various aspects of toxicologic pathology such as anatomic pathology, clinical pathology, experimental pathology, and biomarker research. Publications include society-endorsed Best Practice/Position and Points to Consider publications and ESTP Expert Workshop articles that are relevant to toxicologic pathology and scientific regulatory processes, Opinion articles under the banner of the STP Toxicologic Pathology Forum, Original Articles, Review Articles (unsolicited/contributed, mini, and invited), Brief Communications, Letters to the Editor, Meeting Reports, and Book Reviews. This article provides details on the various publication categories in and will serve as a reference for authors and readers.
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http://dx.doi.org/10.1177/0192623321992305DOI Listing
February 2021

Hallmarks of primary neurulation are conserved in the zebrafish forebrain.

Commun Biol 2021 Jan 29;4(1):147. Epub 2021 Jan 29.

Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD, 21250, USA.

Primary neurulation is the process by which the neural tube, the central nervous system precursor, is formed from the neural plate. Incomplete neural tube closure occurs frequently, yet underlying causes remain poorly understood. Developmental studies in amniotes and amphibians have identified hingepoint and neural fold formation as key morphogenetic events and hallmarks of primary neurulation, the disruption of which causes neural tube defects. In contrast, the mode of neurulation in teleosts has remained highly debated. Teleosts are thought to have evolved a unique mode of neurulation, whereby the neural plate infolds in absence of hingepoints and neural folds, at least in the hindbrain/trunk where it has been studied. Using high-resolution imaging and time-lapse microscopy, we show here the presence of these morphological landmarks in the zebrafish anterior neural plate. These results reveal similarities between neurulation in teleosts and other vertebrates and hence the suitability of zebrafish to understand human neurulation.
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http://dx.doi.org/10.1038/s42003-021-01655-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846805PMC
January 2021

Phosphatidylinositol 3-Kinase δ Inhibitor-Induced Immunomodulation and Secondary Opportunistic Infection in the Cynomolgus Monkey ().

Toxicol Pathol 2020 12 28;48(8):949-964. Epub 2020 Nov 28.

7129Amgen Research, Translational Safety and Bioanalytical Sciences, Thousand Oaks, CA, USA.

Phosphatidylinositol 3-kinases (PI3Ks) regulate intracellular signaling events for multiple cell surface receptors. Phosphatidylinositol 3-kinase δ, 1 of 4 class I PI3K isoforms, is primarily found in leukocytes and regulates immune cell functions. Here, we report changes in the immune and digestive systems that were associated with AMG2519493, a highly selective small-molecule PI3Kδ inhibitor. Following 1- or 3-month oral repeat dosing in the cynomolgus monkey, changes were observed in circulating B cells, lymphoid tissues (spleen, lymph nodes, gut-associated lymphoid tissue, tonsil), and the digestive tract. Decreased circulating B cells and lymphoid cellularity in B cell-rich zones in lymphoid tissues were attributed to the intended pharmacologic activity of AMG2519493. Dose- and duration-dependent digestive system toxicity was characterized by inflammation in the large intestine and secondary opportunistic infections restricted to the digestive tract. Digestive tract changes were associated with moribundity and mortality at high-dose levels, and the effect level decreased with increased duration of exposure. These observations demonstrate the role of PI3Kδ in regulation of the immune system and of host resistance to opportunistic infections of the digestive tract.
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http://dx.doi.org/10.1177/0192623320966238DOI Listing
December 2020

AMG 757, a Half-Life Extended, DLL3-Targeted Bispecific T-Cell Engager, Shows High Potency and Sensitivity in Preclinical Models of Small-Cell Lung Cancer.

Clin Cancer Res 2021 Mar 17;27(5):1526-1537. Epub 2020 Nov 17.

Oncology Research, Amgen Research, Thousand Oaks, California.

Purpose: Small-cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with a high relapse rate, limited therapeutic options, and poor prognosis. We investigated the antitumor activity of AMG 757, a half-life extended bispecific T-cell engager molecule targeting delta-like ligand 3 (DLL3)-a target that is selectively expressed in SCLC tumors, but with minimal normal tissue expression.

Experimental Design: AMG 757 efficacy was evaluated in SCLC cell lines and in orthotopic and patient-derived xenograft (PDX) mouse SCLC models. Following AMG 757 administration, changes in tumor volume, pharmacodynamic changes in tumor-infiltrating T cells (TILs), and the spatial relationship between the appearance of TILs and tumor histology were examined. Tolerability was assessed in nonhuman primates (NHPs).

Results: AMG 757 showed potent and specific killing of even those SCLC cell lines with very low DLL3 expression (<1,000 molecules per cell). AMG 757 effectively engaged systemically administered human T cells, induced T-cell activation, and redirected T cells to lyse tumor cells to promote significant tumor regression and complete responses in PDX models of SCLC and in orthotopic models of established primary lung SCLC and metastatic liver lesions. AMG 757 was well tolerated with no AMG 757-related adverse findings up to the highest tested dose (4.5 mg/kg weekly) in NHP. AMG 757 exhibits an extended half-life in NHP, which is projected to enable intermittent administration in patients.

Conclusions: AMG 757 has a compelling safety and efficacy profile in preclinical studies making it a viable option for targeting DLL3-expressing SCLC tumors in the clinical setting.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2845DOI Listing
March 2021

MRGPRX2 activation as a rapid, high-throughput mechanistic-based approach for detecting peptide-mediated human mast cell degranulation liabilities.

J Immunotoxicol 2020 12;17(1):110-121

Translational Safety & Bioanalytical Sciences, Amgen Research, Amgen, Inc., South San Francisco, CA, USA.

Mast cells play key roles in allergy, anaphylaxis/anaphylactoid reactions, and defense against pathogens/toxins. These cells contain cytoplasmic granules with a wide spectrum of pleotropic mediators that are released upon activation. While mast cell degranulation (MCD) occurs upon clustering of the IgE receptor bound to IgE and antigen, MCD is also triggered through non-IgE-mediated mechanisms, one of which is via Mas-related G protein-coupled receptor X2 (MRGPRX2). MRGPRX2 can be activated by many basic biogenic amines and peptides. Consequently, MRGPRX2-mediated MCD is an important potential safety liability for peptide therapeutics. To facilitate peptide screening for this liability in early preclinical drug development, a rapid, high-throughput engineered CHO-K1 cell-based MRGPRX2 activation assay was evaluated and compared to histamine release in CD34 stem cell-derived mature human mast cells as a reference assay, using 30 positive control and 29 negative control peptides for MCD. Both G protein-dependent (Ca endpoint) and -independent (β-arrestin endpoint) pathways were assessed in the MRGPRX2 activation assay. The MRGPRX2 activation assay had a sensitivity of 100% for both Ca and β-arrestin endpoints and a specificity of 93% (β-arrestin endpoint) and 83% (Ca endpoint) compared to histamine release in CD34 stem cell-derived mature human mast cells. These findings suggest that assessing MRGPRX2 activation in an engineered cell model can provide value as a rapid, high-throughput, economical mechanism-based screening tool for early MCD hazard identification during preclinical safety evaluation of peptide-based therapeutics.
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http://dx.doi.org/10.1080/1547691X.2020.1757793DOI Listing
December 2020

The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity.

Nature 2019 11 30;575(7781):217-223. Epub 2019 Oct 30.

Amgen Research, Amgen Inc, Thousand Oaks, CA, USA.

KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRAS tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRAS tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking.
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http://dx.doi.org/10.1038/s41586-019-1694-1DOI Listing
November 2019

Clinical Implications and Translation of an Off-Target Pharmacology Profiling Hit: Adenosine Uptake Inhibition In Vitro.

Transl Oncol 2019 Oct 19;12(10):1296-1304. Epub 2019 Jul 19.

Immuno-Oncology Therapeutic Area Safety, Global Patient Safety, Labeling and Pediatrics, Amgen Inc., Thousand Oaks, CA, 91320, USA.

Off-target activities of drug candidates observed during in vitro pharmacological profiling frequently do not translate to adverse events (AEs) in human. This could be because off-target activities do not have functional consequences, are not observed at exposures achieved during clinical testing, or may not translate into clinical outcomes. We report clinical consequences of an off-target activity observed during profiling of AMG 337, a selective inhibitor of the mesenchymal-epithelial transition factor being evaluated for treatment of solid tumors. In our screen of 151 potential off-targets, AMG 337 inhibited only adenosine transporter (AT). During clinical trials, headache emerged as the dose-limiting AE in the first-in-human trial. It was thought that headache was caused by extracellular accumulation of adenosine from inhibition of AT by AMG 337 and subsequent adenosine-mediated vasodilation through adenosine receptors (ARs). Further nonclinical studies were performed to evaluate this hypothesis. AMG 337 inhibited AT function in dog and human cells in vitro and dog and human arteries ex vivo. In a dog telemetry study, AMG 337 caused hypotension, which was reduced by pretreatment with theophylline, an AR antagonist. Overall, nonclinical and clinical data suggested that headache was due to cerebral vasorelaxation caused by AMG 337-mediated inhibition of AT. When subjects were advised to drink coffee, an AR antagonist, prior to AMG 337, the severity of headaches was reduced, allowing them to continue treatment. These findings demonstrate the importance of carefully evaluating clinical observations during early drug development and the value of translational nonclinical studies to investigate the mechanism of action driving clinical observations.
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http://dx.doi.org/10.1016/j.tranon.2019.05.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657233PMC
October 2019

Variability of Spleen and Mesenteric Lymph Node in Control Cynomolgus Monkeys ( Macaca fascicularis) from Nonclinical Safety Studies: A Retrospective Assessment.

Toxicol Pathol 2019 01 18;47(1):53-72. Epub 2018 Dec 18.

1 Amgen Inc., South San Francisco, California, USA.

We assessed the variability of spleen and mesenteric lymph node (MLN) microscopic observations and the correlations of these observations with other study data from 478 control cynomolgus monkeys from 53 routine nonclinical safety studies. Spleen weight parameters (absolute and relative to body or brain weights) were highly variable both within a control group on an individual study (up to 5.11-fold) and among animals with the same light microscopic observation. Grades for microscopic observations were also highly variable. The most frequent microscopic observations for spleen were changes in the size and number of germinal centers (58%), acidophilic (hyaline) material in lymphoid follicles (52%), and compound lymphoid follicles (20%). The most frequent microscopic observations in the MLN were eosinophil infiltrates (90%), changes in size and number of germinal centers (42%), and brown pigment (21%). The only meaningful relationships ( r > 0.3) were positive correlations between reticuloendothelial hyperplasia and malarial pigment in the spleen and between each of these observations and spleen weight parameters. We conclude that determination of test article-related effects on the immune system in routine monkey toxicology studies requires careful consideration and a weight-of-evidence approach due to the low numbers of animals/group, the inherent variability in spleen and MLN parameters, and the infrequent correlation among immune system-related end points.
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http://dx.doi.org/10.1177/0192623318809073DOI Listing
January 2019

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo.

J Vis Exp 2017 09 20(127). Epub 2017 Sep 20.

Department of Biological Sciences, University of Maryland, Baltimore County;

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments. The first protocol provides a method to assess the spatial distribution of stable and dynamic MTs and perform a quantitative analysis of these populations with image-processing software. This approach complements existing tools to image microtubule dynamics and distribution in real-time, using transgenic lines or transient expression of tagged constructs. Indeed, such tools are very useful, however they do not readily distinguish between dynamic and stable MTs. The ability to image and analyze these distinct microtubule populations has important implications for understanding mechanisms underlying cell polarization and morphogenesis. The second protocol outlines a technique to analyze nascent MTs specifically. This is accomplished by capturing the de novo growth properties of MTs over time, following microtubule depolymerization with the drug nocodazole and a recovery period after drug washout. This technique has not yet been applied to the study of MTs in zebrafish embryos, but is a valuable assay for investigating the in vivo function of proteins implicated in microtubule assembly.
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http://dx.doi.org/10.3791/55792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752298PMC
September 2017

Development of 2-aminooxazoline 3-azaxanthene β-amyloid cleaving enzyme (BACE) inhibitors with improved selectivity against Cathepsin D.

Medchemcomm 2017 Jun 27;8(6):1196-1206. Epub 2017 Apr 27.

Department of Medicinal Chemistry , Amgen Inc. , One Amgen Center Drive , Thousand Oaks , CA 91320 , USA . Email: ; Tel: +1 805 447 4721.

As part of an ongoing effort at Amgen to develop a disease-modifying therapy for Alzheimer's disease, we have previously used the aminooxazoline xanthene (AOX) scaffold to generate potent and orally efficacious BACE1 inhibitors. While AOX-BACE1 inhibitors demonstrated acceptable cardiovascular safety margins, a retinal pathological finding in rat toxicological studies demanded further investigation. It has been widely postulated that such retinal toxicity might be related to off-target inhibition of Cathepsin D (CatD), a closely related aspartyl protease. We report the development of AOX-BACE1 inhibitors with improved selectivity against CatD by following a structure- and property-based approach. Our efforts culminated in the discovery of a picolinamide-substituted 3-aza-AOX-BACE1 inhibitor absent of retinal effects in an early screening rat toxicology study.
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http://dx.doi.org/10.1039/c7md00106aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072065PMC
June 2017

Maturity-related Variability of the Thymus in Cynomolgus Monkeys (Macaca fascicularis).

Toxicol Pathol 2016 08 25;44(6):874-91. Epub 2016 May 25.

Amgen Inc., Thousand Oaks, California, USA.

Terminal body weights (TBWs), thymus weight parameters, and thymus morphology were retrospectively evaluated in 453 cynomolgus monkeys assigned to control groups on nonclinical toxicity studies. Morphology of bone, ovary, and testis/epididymis were used to determine maturity status of individual animals. There was no correlation between TBW and thymus weight (absolute and/or relative to TBW or brain weight). Thymus weight parameters and grades of decreased lymphocytes in the thymus were highly variable in immature animals compared to mature animals. There was also high (up to 11-fold) variability of thymus weight parameters within a given control group on the same study (generally 3 or 4 animals per sex). Several parameters evaluated had more pronounced age-related changes in males when compared to females. Our results demonstrate the inherent variability of thymus weight parameters and morphologic observations for cynomolgus monkeys on toxicology studies. Changes in thymus parameters in cynomolgus monkeys are unreliable indicators of immunomodulation or immunotoxicity in the absence of other relevant findings. Therefore, the thymus parameters commonly evaluated in preclinical safety assessments should not be the primary data set used to determine the presence of a direct test article-related effect on the immune system.
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http://dx.doi.org/10.1177/0192623316649258DOI Listing
August 2016

In Vitro and In Vivo Activity of AMG 337, a Potent and Selective MET Kinase Inhibitor, in MET-Dependent Cancer Models.

Mol Cancer Ther 2016 07 19;15(7):1568-79. Epub 2016 Apr 19.

Amgen Inc., Thousand Oaks, California.

The MET receptor tyrosine kinase is involved in cell growth, survival, and invasion. Clinical studies with small molecule MET inhibitors have shown the role of biomarkers in identifying patients most likely to benefit from MET-targeted therapy. AMG 337 is an oral, small molecule, ATP-competitive, highly selective inhibitor of the MET receptor. Herein, we describe AMG 337 preclinical activity and mechanism of action in MET-dependent tumor models. These studies suggest MET is the only therapeutic target for AMG 337. In an unbiased tumor cell line proliferation screen (260 cell lines), a closely related analogue of AMG 337, Compound 5, exhibited activity in 2 of 260 cell lines; both were MET-amplified. Additional studies examining the effects of AMG 337 on the proliferation of a limited panel of cell lines with varying MET copy numbers revealed that high-level focal MET amplification (>12 copies) was required to confer MET oncogene addiction and AMG 337 sensitivity. One MET-amplified cell line, H1573 (>12 copies), was AMG 337 insensitive, possibly because of a downstream G12A KRAS mutation. Mechanism-of-action studies in sensitive MET-amplified cell lines demonstrated that AMG 337 inhibited MET and adaptor protein Gab-1 phosphorylation, subsequently blocking the downstream PI3K and MAPK pathways. AMG 337 exhibited potency in pharmacodynamic assays evaluating MET signaling in tumor xenograft models; >90% inhibition of Gab-1 phosphorylation was observed at 0.75 mg/kg. These findings describe the preclinical activity and mechanism of action of AMG 337 in MET-dependent tumor models and indicate its potential as a novel therapeutic for the treatment of MET-dependent tumors. Mol Cancer Ther; 15(7); 1568-79. ©2016 AACR.
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http://dx.doi.org/10.1158/1535-7163.MCT-15-0871DOI Listing
July 2016

Microtubule-associated protein 1b is required for shaping the neural tube.

Neural Dev 2016 Jan 18;11. Epub 2016 Jan 18.

Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD, USA.

Background: Shaping of the neural tube, the precursor of the brain and spinal cord, involves narrowing and elongation of the neural tissue, concomitantly with other morphogenetic changes that contribue to this process. In zebrafish, medial displacement of neural cells (neural convergence or NC), which drives the infolding and narrowing of the neural ectoderm, is mediated by polarized migration and cell elongation towards the dorsal midline. Failure to undergo proper NC results in severe neural tube defects, yet the molecular underpinnings of this process remain poorly understood.

Results: We investigated here the role of the microtubule (MT) cytoskeleton in mediating NC in zebrafish embryos using the MT destabilizing and hyperstabilizing drugs nocodazole and paclitaxel respectively. We found that MTs undergo major changes in organization and stability during neurulation and are required for the timely completion of NC by promoting cell elongation and polarity. We next examined the role of Microtubule-associated protein 1B (Map1b), previously shown to promote MT dynamicity in axons. map1b is expressed earlier than previously reported, in the developing neural tube and underlying mesoderm. Loss of Map1b function using morpholinos (MOs) or δMap1b (encoding a truncated Map1b protein product) resulted in delayed NC and duplication of the neural tube, a defect associated with impaired NC. We observed a loss of stable MTs in these embryos that is likely to contribute to the NC defect. Lastly, we found that Map1b mediates cell elongation in a cell autonomous manner and polarized protrusive activity, two cell behaviors that underlie NC and are MT-dependent.

Conclusions: Together, these data highlight the importance of MTs in the early morphogenetic movements that shape the neural tube and reveal a novel role for the MT regulator Map1b in mediating cell elongation and polarized cell movement in neural progenitor cells.
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http://dx.doi.org/10.1186/s13064-015-0056-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4717579PMC
January 2016

Pharmaceutical Optimization of Peptide Toxins for Ion Channel Targets: Potent, Selective, and Long-Lived Antagonists of Kv1.3.

J Med Chem 2015 Sep 31;58(17):6784-802. Epub 2015 Aug 31.

Therapeutic Discovery, Amgen Inc. , 360 Binney Street, Cambridge, Massachusetts 02142, United States.

To realize the medicinal potential of peptide toxins, naturally occurring disulfide-rich peptides, as ion channel antagonists, more efficient pharmaceutical optimization technologies must be developed. Here, we show that the therapeutic properties of multiple cysteine toxin peptides can be rapidly and substantially improved by combining direct chemical strategies with high-throughput electrophysiology. We applied whole-molecule, brute-force, structure-activity analoging to ShK, a peptide toxin from the sea anemone Stichodactyla helianthus that inhibits the voltage-gated potassium ion channel Kv1.3, to effectively discover critical structural changes for 15× selectivity against the closely related neuronal ion channel Kv1.1. Subsequent site-specific polymer conjugation resulted in an exquisitely selective Kv1.3 antagonist (>1000× over Kv1.1) with picomolar functional activity in whole blood and a pharmacokinetic profile suitable for weekly administration in primates. The pharmacological potential of the optimized toxin peptide was demonstrated by potent and sustained inhibition of cytokine secretion from T cells, a therapeutic target for autoimmune diseases, in cynomolgus monkeys.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00495DOI Listing
September 2015

Development of 2-aminooxazoline 3-azaxanthenes as orally efficacious β-secretase inhibitors for the potential treatment of Alzheimer's disease.

Bioorg Med Chem Lett 2015 Feb 8;25(4):767-74. Epub 2015 Jan 8.

Department of Medicinal Chemistry, Amgen Inc., 360 Binney Street, Cambridge, MA 02142, USA.

The β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is one of the most hotly pursued targets for the treatment of Alzheimer's disease. We used a structure- and property-based drug design approach to identify 2-aminooxazoline 3-azaxanthenes as potent BACE1 inhibitors which significantly reduced CSF and brain Aβ levels in a rat pharmacodynamic model. Compared to the initial lead 2, compound 28 exhibited reduced potential for QTc prolongation in a non-human primate cardiovascular safety model.
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http://dx.doi.org/10.1016/j.bmcl.2014.12.092DOI Listing
February 2015

Retinal Toxicity Induced by a Novel β-secretase Inhibitor in the Sprague-Dawley Rat.

Toxicol Pathol 2015 Jun 31;43(4):581-92. Epub 2014 Oct 31.

Comparative Biology and Safety Sciences, Amgen, Thousand Oaks, California, USA.

β-Secretase 1 (BACE1) represents an attractive target for the treatment of Alzheimer's disease. In the course of development of a novel small molecule BACE1 inhibitor (AMG-8718), retinal thinning was observed in a 1-month toxicity study in the rat. To further understand the lesion, an investigational study was conducted whereby rats were treated daily with AMG-8718 for 1 month followed by a 2-month treatment-free phase. The earliest detectable change in the retina was an increase in autofluorescent granules in the retinal pigment epithelium (RPE) on day 5; however, there were no treatment-related light microscopic changes observed in the neuroretina and no changes observed by fundus autofluorescence or routine ophthalmoscopic examination after 28 days of dosing. Following 2 months of recovery, there was significant retinal thinning attributed to loss of photoreceptor nuclei from the outer nuclear layer. Electroretinographic changes were observed as early as day 14, before any microscopic evidence of photoreceptor loss. BACE1 knockout rats were generated and found to have normal retinal morphology indicating that the retinal toxicity induced by AMG-8718 was likely off-target. These results suggest that AMG-8718 impairs phagolysosomal function in the rat RPE, which leads to photoreceptor dysfunction and ultimately loss of photoreceptors.
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http://dx.doi.org/10.1177/0192623314553804DOI Listing
June 2015

Immunogenicity of Bartonella henselae P26 in cats.

Vet Immunol Immunopathol 2009 Dec 18;132(2-4):251-6. Epub 2009 May 18.

Center for Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of California, One Shields Avenue, Davis, CA 95616, United States.

Cat scratch disease (CSD) has an estimated prevalence of approximately 200,000 persons in the USA, and approximately 22,000 new cases occur annually. Cats are the natural carriers of Bartonella henselae, the agent for CSD. Zoonotic transmission of B. henselae can result in CSD in immunocompetent humans and bacillary angiomatosis in immunosuppressed humans. Infection in cats often goes undetected. Development of a vaccine to prevent feline infection is warranted to reduce the prevalence of infection in the feline population and to decrease the potential for zoonotic transmission. One of the immunoreactive proteins identified from our previous study was P26. In this study, we demonstrated that B. henselae recombinant P26 (rP26) was immunogenic in cats. Four cats immunized with rP26 and four control cats were challenged with B. henselae type I and blood samples were collected for culture, PCR, and serology. Immunization with rP26 did not provide protection against B. henselae infection in cats at the doses used in this study. However, p26 PCR proved to be more sensitive for detection of infection in cats compared to gltA PCR. Furthermore, ELISA using rP26 as the substrate was more sensitive than ELISA using B. henselae type I outer membrane proteins.
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http://dx.doi.org/10.1016/j.vetimm.2009.05.005DOI Listing
December 2009

P26-based serodiagnosis for Bartonella spp. infection in cats.

Comp Med 2008 Aug;58(4):375-80

Center for Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of California, Davis, CA, USA.

Bartonella henselae P26 has been identified as an immunodominant antigen expressed during feline infection. We used antisera from cats experimentally infected with B. henselae (n = 6), B. clarridgeiae (n = 4), or B. koehlerae (n = 2) and from a sample of naturally infected cats (B. henselae, n = 34; B. clarridgeiae, n = 1) to evaluate recombinant P26 (rP26) as a serodiagnostic antigen. Immunoblots using antisera from cats infected with B. henselae and B. clarridgeiae reacted strongly with rP26, whereas B. koehlerae antisera did not. A capture ELISA was designed to evaluate the kinetics of rP26 IgG in sera from experimentally infected cats. For B. henselae and B. clarridgeiae antisera, the kinetic profiles of reactivity were similar for rP26 capture ELISA and Bartonella spp. indirect fluorescence assay. However, for B. koehlerae antisera, reactivity in rP26 capture ELISA was consistently low. The serodiagnostic potential of rP26 capture ELISA was evaluated using sera from cats with known Bartonella sp. exposure histories. All 24 (100%) uninfected cats were seronegative, and 33 of 35 (94.3%) cats bacteremic for Bartonella spp. were seropositive. We propose that rP26-based serology can serve as a useful adjunct tool for the diagnosis of feline infection with B. henselae and B. clarridgeiae, but it may not be useful for feline infection with B. koehlerae.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2706038PMC
August 2008

Genome-wide patterns of single-feature polymorphism in Arabidopsis thaliana.

Proc Natl Acad Sci U S A 2007 Jul 12;104(29):12057-62. Epub 2007 Jul 12.

Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.

We used hybridization to the ATH1 gene expression array to interrogate genomic DNA diversity in 23 wild strains (accessions) of Arabidopsis thaliana (arabidopsis), in comparison with the reference strain Columbia (Col). At <1% false discovery rate, we detected 77,420 single-feature polymorphisms (SFPs) with distinct patterns of variation across the genome. Total and pair-wise diversity was higher near the centromeres and the heterochromatic knob region, but overall diversity was positively correlated with recombination rate (R(2) = 3.1%). The difference between total and pair-wise SFP diversity is a relative measure contrasting diversifying or frequency-dependent selection, similar to Tajima's D, and can be calibrated by the empirical genome-wide distribution. Each unique locus, centered on a gene, has a diversity and selection score that suggest a relative role in past evolutionary processes. Homologs of disease resistance (R) genes include members with especially high levels of diversity often showing frequency-dependent selection and occasionally evidence of a past selective sweep. Receptor-like and S-locus proteins also contained members with elevated levels of diversity and signatures of selection, whereas other gene families, bHLH, F-box, and RING finger proteins, showed more typical levels of diversity. SFPs identified with the gene expression array also provide an empirical hybridization polymorphism background for studies of gene expression polymorphism and are available through the genome browser http://signal.salk.edu/cgi-bin/AtSFP.
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http://dx.doi.org/10.1073/pnas.0705323104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914337PMC
July 2007

Experimental infection of domestic cats with passaged genotype I Bartonella henselae.

Vet Microbiol 2007 Jun 31;122(3-4):290-7. Epub 2007 Jan 31.

Center for Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of California, Davis, CA 95616, USA.

The influence of in vitro passage on Bartonella henselae pathogenesis in cats has not been thoroughly evaluated. Our objective was to examine the bacterial kinetics and humoral immune responses in cats experimentally infected with three different in vitro passages of B. henselae F1, a genotype I strain of feline origin. The F1 strain was in vitro passaged 20 and 40 times, and each was inoculated into a group of 5 cats. The kinetics of bacteremia and the feline humoral immune response to bacterial antigens were compared to a previous study involving a group of six cats inoculated with the original F1 strain. Among the three groups of cats, the kinetics of bacteremia profiles and the humoral immune responses to B. henselae lysates were similar. The influence of passage on bacterial membrane proteins was examined. In vitro passage altered the expression of 4/17 (23.5%) bacterial membrane proteins and 6/15 (40%) bacterial membrane antigens. An association between poor seroreactivity to three lysate antigens (15-, 18- and 45kDa), prolonged bacteremia and decreased serum bactericidal activity was noted. Our data show that in vitro passage of B. henselae did not alter the kinetics of bacteremia, including the occurrence of relapsing bacteremia, in experimentally infected cats. This suggests that highly passaged strains may not be suitable for future vaccination studies. Furthermore, in vitro passage results in phenotypic and antigenic changes in the bacterial membrane protein profile, which warrants caution in the interpretation of studies involving passaged B. henselae strains.
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http://dx.doi.org/10.1016/j.vetmic.2007.01.018DOI Listing
June 2007

Cloning, characterization, and expression of Bartonella henselae p26.

Clin Vaccine Immunol 2006 Aug;13(8):830-6

Center for Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of California at Davis, California 95616, USA.

In order to identify immunoreactive Bartonella henselae proteins, B. henselae antiserum from an experimentally infected cat was used to screen a B. henselae genomic DNA expression library. One immunoreactive phage clone contained a gene (p26) with significant nucleotide identity with orthologs in brucellae, bartonellae, and several plant-associated bacteria. p26 gene sequences from four B. henselae strains, one B. koehlerae strain, and one B. clarridgeiae strain were cloned. Comparative nucleotide sequence analysis showed that p26 is a potential marker for molecular diagnosis of infection, as well as for identification to species level and genotyping of Bartonella sp. isolates. Alignment of the predicted amino acid sequences illustrated conserved putative protein features including a hydrophobic transmembrane region, a peptide cleavage site, and four dominant antigenic sites. Expression of p26 in Escherichia coli produced two proteins (26 and 27.5 kDa), both of which were reactive with feline anti-B. henselae antisera. Furthermore, murine hyperimmune serum raised against either recombinant protein reacted with both proteins. No reactivity to either recombinant protein was detected in nonimmune serum, and reactivity persisted as long as 20 weeks for one cat. The p26 protein product is an immunodominant antigen that is expressed during infection in cats as a preprotein and is subsequently cleaved to form mature P26.
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http://dx.doi.org/10.1128/CVI.00135-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1539124PMC
August 2006

FRIGIDA-independent variation in flowering time of natural Arabidopsis thaliana accessions.

Genetics 2005 Jul 23;170(3):1197-207. Epub 2005 May 23.

Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) are two genes that, unless plants are vernalized, greatly delay flowering time in Arabidopsis thaliana. Natural loss-of-function mutations in FRI cause the early flowering growth habits of many A. thaliana accessions. To quantify the variation among wild accessions due to FRI, and to identify additional genetic loci in wild accessions that influence flowering time, we surveyed the flowering times of 145 accessions in long-day photoperiods, with and without a 30-day vernalization treatment, and genotyped them for two common natural lesions in FRI. FRI is disrupted in at least 84 of the accessions, accounting for only approximately 40% of the flowering-time variation in long days. During efforts to dissect the causes for variation that are independent of known dysfunctional FRI alleles, we found new loss-of-function alleles in FLC, as well as late-flowering alleles that do not map to FRI or FLC. An FLC nonsense mutation was found in the early flowering Van-0 accession, which has otherwise functional FRI. In contrast, Lz-0 flowers late because of high levels of FLC expression, even though it has a deletion in FRI. Finally, eXtreme array mapping identified genomic regions linked to the vernalization-independent, late-flowering habit of Bur-0, which has an alternatively spliced FLC allele that behaves as a null allele.
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http://dx.doi.org/10.1534/genetics.104.036533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1451178PMC
July 2005

Quantitative trait locus mapping and DNA array hybridization identify an FLM deletion as a cause for natural flowering-time variation.

Proc Natl Acad Sci U S A 2005 Feb 4;102(7):2460-5. Epub 2005 Feb 4.

Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.

Much of the flowering time variation in wild strains of Arabidopsis thaliana is due to allelic variation at two epistatically acting loci, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). FLC encodes a MADS (MCM1/AGAMOUS/DEFICIENS/SRF1) domain transcription factor that directly represses a series of flowering-promoting genes. FRI and FLC, however, do not explain all of the observed variation, especially when plants are grown in short days. To identify loci that act in addition to FRI and FLC in controlling flowering of natural accessions, we have analyzed a recombinant inbred line population derived from crosses of accession Niederzenz (Nd) to Columbia, both of which contain natural FRI lesions. Quantitative trait locus mapping and genomic DNA analysis by microarray hybridization were used to identify candidate genes affecting variation in flowering behavior. In both long and short days, the quantitative trait locus of largest effect, termed FLOWERING 1 (FLW1), was found to be associated with a Nd-specific deletion of FLOWERING LOCUS M (FLM), which encodes a floral repressor closely related to FLC. Analysis of near isogenic lines and quantitative transgenic complementation experiments confirmed that the FLM deletion is, in large part, responsible for the early flowering of the Nd accession.
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http://dx.doi.org/10.1073/pnas.0409474102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC548991PMC
February 2005

Haplotype structure and phenotypic associations in the chromosomal regions surrounding two Arabidopsis thaliana flowering time loci.

Genetics 2004 Nov;168(3):1627-38

Molecular and Computational Biology Program, University of Southern California, Los Angeles, California 90089, USA.

The feasibility of using linkage disequilbrium (LD) to fine-map loci underlying natural variation in Arabidopsis thaliana was investigated by looking for associations between flowering time and marker polymorphism in the genomic regions containing two candidate genes, FRI and FLC, both of which are known to contribute to natural variation in flowering. A sample of 196 accessions was used, and polymorphism was assessed by sequencing a total of 17 roughly 500-bp fragments. Using a novel Bayesian algorithm based on haplotype similarity, we demonstrate that LD could have been used to fine-map the FRI gene to a roughly 30-kb region and to identify two common loss-of-function alleles. Interestingly, because of genetic heterogeneity, simple single-marker associations would not have been able to map FRI with nearly the same precision. No clear evidence for previously unknown alleles at either locus was found, but the effect of population structure in causing false positives was evident.
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http://dx.doi.org/10.1534/genetics.104.029470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1448803PMC
November 2004

Management of hemodynamic changes associated with removal of a large abdominal myofibroblastic tumor in a pony.

J Am Vet Med Assoc 2004 Oct;225(7):1079-83, 1049

Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.

A 22-year-old female Welsh-cross pony was evaluated because of intermittent colic, signs of depression, pyrexia, anorexia, muscle wasting with abdominal distention, and weight gain over the preceding 12 months. A large abdominal mass was detected and surgically removed; the hemodynamic alterations and complications caused by the dramatic fluid losses and shifts that can occur in association with removal of a large abdominal mass required extensive postoperative management. Monitoring of clinical and hematologic variables such as attitude, heart rate, mucous membrane color, mean arterial blood pressure, PCV, and plasma total protein concentration provided useful information for successful management of the patient after surgery. On removal, the tumor weighed 19% of the pony's body weight and was characterized as a myofibroblastic tumor. Myofibroblastic tumors should be considered as a differential for large internal abdominal masses in horses, and surgical removal may be feasible and life extending with appropriate postoperative care.
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http://dx.doi.org/10.2460/javma.2004.225.1079DOI Listing
October 2004

Biologic behavior and prognostic factors for mast cell tumors of the canine muzzle: 24 cases (1990-2001).

J Vet Intern Med 2003 Sep-Oct;17(5):687-92

Veterinary Medical Teaching Hospital, University of California, School of Veterinary Medicine, Davis, CA, USA.

The medical records of 24 dogs with histologically confirmed mast cell tumors (MCT) of the muzzle were retrospectively evaluated to determine their biologic behavior and prognostic factors. Information on signalment, tumor grade and stage, treatment methods, and pattern of and time to failure and death was obtained from the medical record. Twenty-three dogs were treated with combinations of radiotherapy, surgery, and chemotherapy; 1 dog received no treatment. There were 2 Grade 1, 15 Grade 11, and 7 Grade III tumors. Tumors were stage 0 (n = 8), stage 1 (5), stage 2 (6), stage 3 (4), and stage 4 (1). Mean and median survival times of treated dogs were 36 and 30 months, respectively. Prognostic factors affecting survival time included tumor grade and presence of metastasis at diagnosis. Dogs with Grade I and II tumors survived longer than dogs with Grade III tumors. Variables, including sex, age, gross versus microscopic disease, and treatment type were not found to affect survival. Local control rate was 75% at 1 year and 50% at 3 years. Tumor grade was the only variable found to affect local control. Dogs with Grade I tumors had longer disease-free intervals than those with Grade II tumors, and dogs with Grade II tumors had longer disease-free intervals than dogs with Grade III tumors. Eight of 9 dogs dying of MCT had local or regional disease progression. Muzzle MCT a rebiologically aggressive tumors with higher regional metastatic rates than previously reported for MCT in other sites.
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http://dx.doi.org/10.1111/j.1939-1676.2003.tb02501.xDOI Listing
December 2003

Evaluation of a collagen patch as a method of enhancing ventriculotomy healing in Japanese quail (Coturnix coturnix japonica).

Vet Surg 2003 Mar-Apr;32(2):103-12

Veterinary Medical Teaching Hospital, and the Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis 95616, USA.

Objective: To assess the ability of a porcine submucosal collagen patch to enhance ventriculotomy healing in quail.

Study Design: Histologic assessment of wound repair after ventriculotomy. Animal Population-Eighteen adult Japanese quail.

Methods: All quail had a ventriculotomy through the caudoventral thin muscle. The quail were randomly assigned to 1 of 2 groups: group I (n = 9) had routine closure and group II (n = 9) had routine closure and the application of a porcine submucosal collagen patch over the serosal surface of the ventricular suture line. Three quail from each group underwent necropsy at 7, 14, and 21 days after surgery and healing of the ventriculotomy site was evaluated by microscopy.

Results: Ventricular mucosal epithelium was completely healed at 21 days postoperatively. Time to restoration of the ventricular submucosal integrity was variable in both groups. There was evidence of a gross or microscopic perforation in 4 quail in group II, which was statistically significant (P =.041). Quail with perforations had significantly elevated numbers of ventricular serosal lymphoid aggregates compared with those without perforations (P =.029). There were no other significant temporal differences between group I and group II for mortality or histopathologic grading of wound healing.

Conclusions: A porcine collagen patch did not enhance ventriculotomy wound healing. Subjectively, the collagen patch might have contributed to perforation by the generation of a lymphocytic xenograft rejection response.

Clinical Relevance: Ventriculotomy can be safely performed using routine closure methods in healthy birds.
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http://dx.doi.org/10.1053/jvet.2003.50011DOI Listing
May 2003

Accuracy of magnetic resonance imaging for estimating intramedullary osteosarcoma extent in pre-operative planning of canine limb-salvage procedures.

Vet Radiol Ultrasound 2002 Sep-Oct;43(5):432-41

Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California, Davis 95616, USA.

The objective of this work was to compare the accuracy of radiographs and magnetic resonance imaging (MRI) for estimating appendicular osteosarcoma margins. The accuracy of computed tomography (CT) and bone scintigraphy was also assessed when these studies were available. Eight dogs with appendicular osteosarcoma underwent radiographic and MRI of affected limbs. In addition, bone scintigraphy was performed in six dogs and CT examination was performed in five dogs. Two observers jointly measured tumor length on all imaging studies. Correlative gross and histologic evaluation of all affected limbs was performed to determine tumor extent as measured from the nearest articular surface. Results from imaging studies were compared to gross and microscopic morphometry findings to determine the accuracy of each modality for determining tumor boundaries. MRI images were accurate with a mean overestimation of actual tumor length of 3 +/- 13%. T1-weighted non-contrast images were superior in identifying intramedullary tumor margins in most instances whereas contrast-enhanced images provided supplemental information in two dogs. Lateromedial and craniocaudal radiographs overestimated tumor length by 17 +/- 28% and 4 +/- 26%, respectively. Scintigraphy and CT overestimated tumor margins by 14 +/- 28% and 27 +/- 36%, respectively. MRI appears to be an accurate diagnostic imaging modality in determining intramedullary osteosarcoma boundaries. MRI should be considered as part of a pre-operative assessment of appendicular osteosarcoma, particularly when a limb-sparing procedure is contemplated.
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http://dx.doi.org/10.1111/j.1740-8261.2002.tb01030.xDOI Listing
February 2003

Quantitative trait loci controlling light and hormone response in two accessions of Arabidopsis thaliana.

Genetics 2002 Feb;160(2):683-96

Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

We have mapped quantitative trait loci (QTL) responsible for natural variation in light and hormone response between the Cape Verde Islands (Cvi) and Landsberg erecta (Ler) accessions of Arabidopsis thaliana using recombinant inbred lines (RILs). Hypocotyl length was measured in four light environments: white, blue, red, and far-red light and in the dark. In addition, white light plus gibberellin (GA) and dark plus the brassinosteroid biosynthesis inhibitor brassinazole (BRZ) were used to detect hormone effects. Twelve QTL were identified that map to loci not previously known to affect light response, as well as loci where candidate genes have been identified from known mutations. Some QTL act in all environments while others show genotype-by-environment interaction. A global threshold was established to identify a significant epistatic interaction between two loci that have few main effects of their own. LIGHT1, a major QTL, has been confirmed in a near isogenic line (NIL) and maps to a new locus with effects in all light environments. The erecta mutation can explain the effect of the HYP2 QTL in the blue, BRZ, and dark environments, but not in far-red. LIGHT2, also confirmed in an NIL, has effects in white and red light and shows interaction with GA. The phenotype and map position of LIGHT2 suggest the photoreceptor PHYB as a candidate gene. Natural variation in light and hormone response thus defines both new genes and known genes that control light response in wild accessions.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1461994PMC
February 2002