Lukas C.,Lapeyronie Hospital |
Van Der Heijde D.,Leiden University |
Fatenajad S.,Wyeth Research
Annals of the Rheumatic Diseases | Year: 2010
Background: Negative radiographic change scores obtained under blinded time-sequence conditions suggest that repair of joints may indeed occur. It is likely that, if repair truly exists, it would be preferentially seen in clinically inactive joints from patients treated with drugs with well-known structural efficacy. Objective: To determine whether repair is associated with both the absence or improvement of swelling and with treatment. Patients and methods: Radiographs from patients of the TEMPO trial were scored twice by two readers according to the Sharp-van der Heijde score, blinded to both treatment and true time sequence. Single-joint change scores in erosions were coupled with single-joint swelling scores obtained from clinical examination. Consistency of observed improvement across readers and repeat reads was described, and factors expected to increase the likelihood of occurrence of both worsening and improvement of erosion were tested by generalised estimating equations (GEE) modelling. Results: In all of the four independent reads, the mean change in erosion score was statistically significantly negative only in the subgroup of joints with absent or improved swelling, when erosions were present at baseline. Multivariate analysis showed that worsening of the erosion score in a joint was significantly increased if that joint was already damaged at study entry, clinical swelling persisted and methotrexate was used instead of etanercept. Repair was associated with improvement of swelling and use of etanercept (p≤0.007 for all associations). Conclusion: Repair of erosions almost exclusively occurs in joints with improvement or absence of swelling, in patients treated with etanercept. Progression is seen more frequently in joints with persistent swelling, in patients receiving methotrexate monotherapy, primarily if damage is already present.
Mazumdar J.,University of Pennsylvania |
Mazumdar J.,Howard Hughes Medical Institute |
Mazumdar J.,Glaxosmithkline |
O'Brien W.T.,University of Pennsylvania |
And 6 more authors.
Nature Cell Biology | Year: 2010
Stem cells reside in specialized microenvironments or 'niches' that regulate their function. In vitro studies using hypoxic culture conditions (< 5% O2) have revealed strong regulatory links between O2 availability and functions of stem and precursor cells. Although some stem cells are perivascular, others may occupy hypoxic niches and be regulated by O 2 gradients. However, the underlying mechanisms remain unclear. Here, we show that hypoxia inducible factor-1 ± (HIF-1 ±), a principal mediator of hypoxic adaptations, modulates Wnt/β-catenin signalling in hypoxic embryonic stem (ES) cells by enhancing β-catenin activation and expression of the downstream effectors LEF-1 and TCF-1. This regulation extends to primary cells, including isolated neural stem cells (NSCs), and is not observed in differentiated cells. In vivo, Wnt/β-catenin activity is closely associated with low O2 regions in the subgranular zone of the hippocampus, a key NSC niche. Hif-1 ± deletion impairs hippocampal Wnt-dependent processes, including NSC proliferation, differentiation and neuronal maturation. This decline correlates with reduced Wnt/β-catenin signalling in the subgranular zone. O2 availability, therefore, may have a direct role in stem cell regulation through HIF-1 ± modulation of Wnt/β-catenin signalling. © 2010 Macmillan Publishers Limited. All rights reserved.
Rahimi E.,Islamic Azad University |
Ameri M.,Wyeth Research
Food Control | Year: 2011
This study was conducted to determine the prevalence and antimicrobial resistance pattern of Campylobacter spp. isolated from retail raw poultry meats in Iran. From July 2009 to March 2010, a total of 494 raw meat samples from chicken (n = 200), turkey (n = 170), quail (n = 86), partridge (n = 17), and ostrich (n = 21) were purchased from randomly selected retail outlets in Shahrekord, Iran. Using cultural method, 187 meat samples (37.9%) were contaminated with Campylobacter. The highest prevalence of Campylobacter spp. was found in chicken meat (47.0%) followed by quail (43.0%), partridge (35.3%), turkey (28.8%), and ostrich (4.8%) meat. The most prevalent Campylobacter species was Campylobacter jejuni (92.0%). The PCR assay could identify 38 Campylobacter-contaminated samples that were negative using the cultural method. Antimicrobial susceptibility test results showed that 98.4% of isolates were resistant to one or more antimicrobial agents. Resistance to tetracycline was the most common findings (70.6%), followed by resistance to nalidixic acid (54.0%), and ciprofloxacin (49.7%). Significantly higher prevalence rates of Campylobacter spp. (P < 0.05) were found in meat samples taken in summer (51.1%). To our knowledge, the present study is the first report of the isolation of Campylobacter spp. from raw partridge meat in Iran. © 2011 Elsevier Ltd.
Hao W.,Wyeth Research |
Chang C.-P.B.,Wyeth Research |
Tsao C.-C.,Wyeth Research |
Xu J.,Wyeth Research
Journal of Biological Chemistry | Year: 2010
Cancer cells constantly adapt to oxidative phosphorylation (OXPHOS) suppression resulting from hypoxia or mitochondria defects. Under the OXPHOS suppression, AMP-activated protein kinase (AMPK) regulates global metabolism adjustments, but its activation has been found to be transient. Whether cells can maintain cellular ATP homeostasis and survive beyond the transient AMPK activation is not known. Here, we study the bioenergetic adaptation to the OXPHOS inhibitor oligomycin in a group of cancer cells.Wefound that oligomycin at 100 ng/ml completely inhibits OXPHOS activity in 1 h and induces various levels of glycolysis gains by 6 h, from which we calculate the bioenergetic organizations of cancer cells. In glycolysis-dominant cells, oligomycin does not induce much energy stress as measured by glycolysis acceleration, ATP imbalance, AMPKactivation,AMPKsubstrate acetyl-CoA carboxylase phosphorylation at Ser79, and cell growth inhibition. In OXPHOS-dependent LKB1 wild type cells, oligomycin induces 5-8% ATP drops and transient AMPK activation during the initial 1-2 h. After AMPK activation is completed, oligomycininduced increase of acetyl-CoA carboxylase phosphorylation at Ser79 is still detected, and cellular ATP is back at preoligomycin treatment levels by sustained elevation of glycolysis. Cell growth, however, is inhibited without an increase in cell death and alteration in cell cycle distribution. In OXPHOS-dependent LKB1-null cells, no AMPK activation by oligomycin is detected, yet cells still show a similar adaptation. We also demonstrate that the adaptation to oligomycin does not invoke activation of hypoxia-induced factor. Our data suggest that cancer cells may grow and survive persistent OXPHOS suppression through an as yet unidentified regulatory mechanism. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
Wang X.,Wyeth Research
Advances in clinical chemistry | Year: 2010
In cardiovascular disease rupture of a vulnerable atherosclerotic plaque is the major causative factor of acute coronary syndromes, myocardial infarction and stroke, and can ultimately lead to death. Identification of biomarkers that could accurately predict the risk of plaque rupture would be a significant advance in guiding treatment of patients with this disease, The use of these biomarkers would also facilitate the development of new drugs to treat cardiovascular disease, particularly those that act through novel mechanisms. There is currently a lack of specific biomarkers for vulnerable plaque, and thus, it is an area of intense research including the concepts of live detection versus retrospective characterization, molecular imaging, and biochemical biomarker discovery. This review will focus on recent advances on both imaging and circulating molecular biomarkers in atherosclerosis. The use of combinations of different imaging modalities (such as molecular, functional, and anatomical) and modalities with circulating/biochemical markers is the current trend and will likely provide the most useful information for the assessment of the vulnerability of atherosclerotic plaques.
Brooijmans N.,Wyeth Research |
Humblet C.,Wyeth Research
Journal of Computer-Aided Molecular Design | Year: 2010
Virtual screening has become a popular tool to identify novel leads in the early phases of drug discovery. A variety of docking and scoring methods used in virtual screening have been the subject of active research in an effort to gauge limitations and articulate best practices. However, how to best utilize different scoring functions and various crystal structures, when available, is not yet well understood. In this work we use multiple crystal structures of PI3 K-γ in both prospective and retrospective virtual screening experiments. Both Glide SP scoring and Prime MM-GBSA rescoring are utilized in the prospective and retrospective virtual screens, and consensus scoring is investigated in the retrospective virtual screening experiments. The results show that each of the different crystal structures that was used, samples a different chemical space, i.e. different chemotypes are prioritized by each structure. In addition, the different (re)scoring functions prioritize different chemotypes as well. Somewhat surprisingly, the Prime MM-GBSA scoring function generally gives lower enrichments than Glide SP. Finally we investigate the impact of different ligand preparation protocols on virtual screening enrichment factors. In summary, different crystal structures and different scoring functions are complementary to each other and allow for a wider variety of chemotypes to be considered for experimental follow-up. © 2010 Springer Science+Business Media B.V.
Liu K.,Wyeth Research
Assay and drug development technologies | Year: 2010
ABSTRACT Transient receptor potential (TRP) channels have been found to play important roles in cellular physiology and hold promise as therapeutic targets. These channels activate in response to a variety of chemical or physical stimuli and conduct non-selective cation currents (NSCC). Due to their unique activation properties, application of automated electrophysiology to measure the channel activity has been difficult. Using HEK293 cells stably expressing human TRP channels, hTRPC6 and hTRPA1, we developed and validated a high-throughput Rb(+) efflux assay for NSCC channels. The assay was performed in cell-based 96-well format. A significant increase in Rb(+) efflux can be detected upon channel activation by specific agonists, confirming that both TRPC6 and TRPA1 channels are permeable to Rb(+) ions. The agonists induced Rb(+) efflux can be blocked by known channel blockers and selected compounds from our high-throughput screening (HTS) hits. The assay is suitable for HTS with Z' factors of 0.53 and above. We also tested the Ca(2+) effect on channel activities in this assay. Both TRPC6 and TRPA1 channels were found to be inhibited by increasing the concentration of Ca(2+) in the assay buffer. However, Ca(2+) significantly reduced the potency of allyl isothiocyanate (AITC) on TRPA1 but did not affect the potency of carbochol on TRPC6. Using this assay for secondary confirmation screen, we successfully identified and confirmed the positive hits as TRPC6 inhibitors.
Huang S.,Wyeth Research
Journal of Computational Biology | Year: 2010
High content imaging (HCI) is an increasingly important method for elucidating changes in cellular biology. The combination of validated immuno-fluorescent assays, availability of automated microscopy, and advances in image analysis software has led to increased exploitation of this technology in a variety of settings ranging from cellular signaling pathways to drug discovery. Recent advances in HCI have made the collection of multi-parametric datasets from high-throughput screening routine, but analysis of these data remains a challenge. The few existing analytical tools used to analyze HCI data, usually provided by HCI platform vendors, require extensive operator interaction and, more importantly, lack statistical power. This results in serious under-utilization of the information available from this powerful technology. As HCI applications become increasingly complex, measurements to estimate the composition of the cell population and thus the underlying data structure also becomes more complicated, and the analysis to facilitate the understanding of the biological system will rely heavily on analytical expertise that requires advanced statistical training. The aim of this article is to review the major statistical issues in HCI data analysis, with a focus on quantitative analysis of cell sub-populations. Copyright 2010, Mary Ann Liebert, Inc.
Dagan R.,Soroka University Medical Center |
Givon-Lavi N.,Soroka University Medical Center |
Greenberg D.,Soroka University Medical Center |
Fritzell B.,Wyeth Research |
Siegrist C.-A.,University Hospitals Geneva Medical Center
Journal of Infectious Diseases | Year: 2010
Background. The antibody response to pneumococcal conjugate vaccines (PCVs) in infants is variable. Factors responsible for this variability have not been fully elucidated. The objective of this study was to investigate whether pneumococcal carriage around the time of the first dose of 7-valent PCV (PCV7) affects serotype-specific immunologic response. Methods. Healthy 2-month old infants were randomized to receive 2 (at the ages of 4 and 6 months) or 3 (at the ages of 2, 4, and 6 months) PCV7 doses and a booster dose (at the age of 12 months). Nasopharyngeal or oropharyngeal specimens were obtained for culture shortly before the first PCV7 dose. Serotype-specific immunoglobulin (Ig) G levels were measured at ages 2, 7, and 13 months. Results. Of 545 children studied, 332 received a booster dose. The most common serotypes carried around the time of the first PCV7 dose were 6B (n = 37), 19F (n = 22), and 23F (n = 14). In carriers before the first dose, the IgG response to the carried serotype after 2 or 3 doses was significantly lower than in noncarriers. In contrast, response to the noncarried serotypes was not affected. Although all children responded to the booster dose, the response to the originally carried serotype was generally lower. Conclusions. Serotype-specific hyporesponsiveness to PCV7 after pneumococcal carriage in infants is demonstrated for the first time. This phenomenon was common, lasted for at least several months, and was only partially overcome by the 12-month booster. Trial registration. isrctn.org identifier: ISRCTN28445844. © 2010 by the Infectious Diseases Society of America. All rights reserved.
Wu Y.,Columbia University |
Yanase E.,Columbia University |
Feng X.,Wyeth Research |
Siegel M.M.,Wyeth Research |
Sparrow J.R.,Columbia University
Proceedings of the National Academy of Sciences of the United States of America | Year: 2010
Fluorescent bisretinoids, such as A2E and all-trans-retinal dimer, form as a by-product of vitamin A cycling in retina and accumulate in retinal pigment epithelial (RPE) cells as lipofuscin pigments. These pigments are implicated in pathological mechanisms involved in several vision-threatening diseases including age-related macular degeneration. Efforts to understand damaging events initiated by these bisretinoids have revealed that photoexcitation of A2E by wavelengths in the visible spectrum leads to singlet oxygen production and photooxidation of A2E. Here we have employed liquid chromatography coupled to electrospray ionization mass spectrometry together with tandem mass spectrometry (MS/MS), to demonstrate that A2E also undergoes photooxidation-induced degradation and we have elucidated the structures of some of the aldehyde-bearing cleavage products. Studies in which A2E was incubated with a singlet oxygen generator yielded results consistent with a mechanism involving bisretinoid photocleavage at sites of singlet molecular oxygen addition. We provide evidence that one of the products released by A2E photodegradation is methylglyoxal, a low molecular weight reactive dicarbonyl with the capacity to form advanced glycation end products. Methylglyoxal is already known to be generated by carbohydrate and lipid oxidation; this is the first report of its production via bisretinoid photocleavage. It is significant that AGE-modified proteins are detected in deposits (drusen) that accumulate below RPE cells in vivo; drusen have been linked to age-related macular degeneration pathogenesis. Whereas various processes play a role in drusen formation, these findings are indicative of a contribution from lipofuscin photo-oxidation in RPE.