66 research outputs found
Protein Microarray Analysis of Disease Activity in Pediatric Inflammatory Bowel Disease Demonstrates Elevated Serum PLGF, IL-7, TGF-beta1, and IL-12p40 Levels in Crohn's Disease and Ulcerative Colitis Patients in Remission versus Active Disease
OBJECTIVES—Cytokines and growth factors play a major role in the dysregulated immune response in inflammatory bowel disease (IBD). We hypothesized that significant differences exist between the serum cytokine and growth factor profiles of pediatric IBD patients with active disease (AD) and those in remission, and that levels of some of these soluble mediators may be used to define regulators in IBD and determine disease activity. METHODS—Eighty-eight, consecutive patients with confirmed Crohn’s disease (CD) and ulcerative colitis (UC) seen at the Duke Children’s Hospital were prospectively enrolled and a serum sample was obtained. Data were recorded at the time of serum collection to calculate disease activity indices. The relative expression of 78 cytokines, growth factors, and soluble receptors was determined using proprietary antibody-based protein microarrays amplified by rolling circle amplification. SPSS 8 (SPSS Inc., Chicago, IL) was used to compare protein profiles for CD and UC patients in clinical remission (CR) versus AD. RESULTS—Sixty-five CD patients and 23 UC patients were enrolled. Forty-one CD patients had available samples and PCDAI results. Twenty-two patients were in remission PCDAI ≤ 12.5 (median 5), 19 patients had disease activity >15 (median 30). Univariate analysis revealed that PLGF, IL-7, IL-12p40, and TGF-β1 cytokine levels were significantly elevated for patients in CR versus AD (p 110. Only one cytokine, IL12p40, showed significance between CR versus AD (p < 0.02). CONCLUSIONS—Surprisingly, we found no differences in circulating levels of proinflammatory cytokines but found that pediatric IBD patients in remission compared to those with AD had higher levels of specific circulating cytokines, including the regulatory cytokines IL-12p40, and TGF-β1. It may be that these cytokines directly regulate intestinal inflammation in IBD or reflect the activity of T regulatory cells in negatively regulating the inflammatory response. Further studies will be needed to validate our results to define the molecular pathways involved in the intestinal immune response in man
Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics
BACKGROUND: Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. RESULTS: Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. CONCLUSION: The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings
Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics
Background Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. Results Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. Conclusion The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings
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Clinical validation of a genetic model to estimate the risk of developing choroidal neovascular age-related macular degeneration
Predictive tests for estimating the risk of developing late-stage neovascular age-related macular degeneration (AMD) are subject to unique challenges. AMD prevalence increases with age, clinical phenotypes are heterogeneous and control collections are prone to high false-negative rates, as many control subjects are likely to develop disease with advancing age. Risk prediction tests have been presented previously, using up to ten genetic markers and a range of self-reported non-genetic variables such as body mass index (BMI) and smoking history. In order to maximise the accuracy of prediction for mainstream genetic testing, we sought to derive a test comparable in performance to earlier testing models but based purely on genetic markers, which are static through life and not subject to misreporting. We report a multicentre assessment of a larger panel of single nucleotide polymorphisms (SNPs) than previously analysed, to improve further the classification performance of a predictive test to estimate the risk of developing choroidal neovascular (CNV) disease. We developed a predictive model based solely on genetic markers and avoided inclusion of self-reported variables (eg smoking history) or non-static factors (BMI, education status) that might otherwise introduce inaccuracies in calculating individual risk estimates. We describe the performance of a test panel comprising 13 SNPs genotyped across a consolidated collection of four patient cohorts obtained from academic centres deemed appropriate for pooling. We report on predictive effect sizes and their classification performance. By incorporating multiple cohorts of homogeneous ethnic origin, we obtained >80 per cent power to detect differences in genetic variants observed between cases and controls. We focused our study on CNV, a subtype of advanced AMD associated with a severe and potentially treatable form of the disease. Lastly, we followed a two-stage strategy involving both test model development and test model validation to present estimates of classification performance anticipated in the larger clinical setting. The model contained nine SNPs tagging variants in the regulators of complement activation (RCA) locus spanning the complement factor H (CFH), complement factor H-related 4 (CFHR4), complement factor H-related 5 (CFHR5) and coagulation factor XIII B subunit (F13B) genes; the four remaining SNPs targeted polymorphisms in the complement component 2 (C2), complement factor B (CFB), complement component 3 (C3) and age-related maculopathy susceptibility protein 2 (ARMS2) genes. The pooled sample size (1,132 CNV cases, 822 controls) allowed for both model development and model validation to confirm the accuracy of risk prediction. At the validation stage, our test model yielded 82 per cent sensitivity and 63 per cent specificity, comparable with metrics reported with earlier testing models that included environmental risk factors. Our test had an area under the curve of 0.80, reflecting a modest improvement compared with tests reported with fewer SNPs
The human telomeric protein hTRF1 induces telomere-specific nucleosome mobility
Human telomeres consist of thousands of base pairs of double-stranded TTAGGG repeats, organized by histone proteins into tightly spaced nucleosomes. The double-stranded telomeric repeats are also specifically bound by the telomeric proteins hTRF1 and hTRF2, which are essential for telomere length maintenance and for chromosome protection. An unresolved question is what role nucleosomes play in telomere structure and dynamics and how they interact and/or compete with hTRF proteins. Here we show that hTRF1 specifically induces mobility of telomeric nucleosomes. Moreover, Atomic Force Microscopy (AFM) imaging shows that hTRF1 induces compaction of telomeric DNA only in the presence of a nucleosome, suggesting that this compaction occurs through hTRF1–nucleosome interactions. Our findings reveal an unknown property of hTRF1 that has implications for understanding telomere structure and dynamics
The Telomere Binding Protein TRF2 Induces Chromatin Compaction
Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE) studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures
Organization of mammalian telomere chromatin.
Telomeres are specialized nucleoprotein structures at the ends of eukaryotic chromosomes. They protect ends of chromosomes from fusion and degradation. The telomere DNA of most eukaryotes consists of stretches of simple repetitive DNA sequences with G and C rich strands. Telomeres from the many somatic cell lines are shortened with successive cell divisions. The shortening of telomeres is associated with aging and senescence and can serve as a molecular clock to prevent oncogenesis. Telomere length can be stabilized by telomerase, the reverse transcriptase that adds the telomeric repeats at the ends of chromosomes. Telomere nucleoprotein of low eukaryotes such as yeast is devoid of nucleosomes and organized as a special telomere-specific structure called the telosome, which is involved in telomere length regulation and protection. However, the organization of mammalian telomere nucleoprotein was not known. In this dissertation I have used biochemical and biophysical techniques to investigate the structure of mammalian telomere nucleoprotein. Techniques used include conventional nuclease probing of the structural features of telomere nucleoprotein as well as three novel techniques developed by me: (1) isolation of formaldehyde-crosslinked rat telomere nucleoprotein, (2) femtosecond UV laser crosslinking of telomere nucleoprotein inside of human nuclei, (3) mapping of the location and distribution of telomere proteins with respect to the ends of the telomere. We have shown that mammalian telomeres are organized as telomere-specific chromatin with closely spaced nucleosomes about 40 bp shorter than nucleosomes of bulk chromatin. In contrast to the situation in lower eukaryotes, nucleosomes compromise a major part of metazoan telomeres. However, nucleosomes are absent at the last kilobase of human telomeres. The protein TRF1 is present at the end of telomeres and interspersed with nucleosomes at the internal telomere regions. TRF2 is localized and binds to the last 600 bp of telomeres. We have proposed that human telomeres have telosome like structure at the last 1 kb.Ph.D.BiochemistryBiological SciencesBiophysicsMolecular biologyPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/131706/2/9929879.pd
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