[Show abstract][Hide abstract] ABSTRACT: This study aimed to reliably identify serum protein profile alterations that may be useful for elucidation of the disease mechanism and/or finding new targets for treatment and intervention.
International journal of gynecological cancer : official journal of the International Gynecological Cancer Society. 06/2014;
[Show abstract][Hide abstract] ABSTRACT: There is tremendous scientific and clinical value to further improve the predictive power of autoantibodies as autoantibody-positive (AbP) children have heterogeneous rates of progression to clinical diabetes. This study explored the potential of using gene expression profiles as biomarkers for risk stratification among 104 AbP subjects from the Diabetes Autoimmunity Study in the Young (DAISY) using a discovery dataset based on microarray and a validation dataset based on Real-Time RT-PCR. Our microarray data identified 454 candidate genes with expression levels that are associated with different T1D progression rates. RT-PCR analyses of the top 27 candidate genes confirmed five genes (BACH2, IGLL3, EIF3A, CDC20 and TXNDC5) that are associated with differential progression and implicated in lymphocyte activation and function. Multivariate analyses of these five genes in the discovery and validation datasets identified and confirmed four multi-gene models (BI, ICE, BICE and BITE, each letter representing a gene) that consistently stratify high and low risk subsets of AbP subjects with hazard ratios (HR) greater than 6 (p < 0.01). These results suggest that these genes may be involved in T1D pathogenesis and potentially serve as excellent gene expression biomarkers to predict the risk of progression to clinical diabetes for AbP subjects.
[Show abstract][Hide abstract] ABSTRACT: A novel monoclonal antibody (mAb), known as AC10364, was identified from an antibody library generated by immunization of mice with human carcinoma cells. The mAb recognized proteins in lysates from multiple carcinoma cell lines. Cell cytotoxicity assays showed that AC10364 significantly inhibited cell growth and induced apoptosis in multiple carcinoma cell lines, including Bel/fu, KATO-III and A2780. Compared with mAb AC10364 or chemotherapeutic drugs alone, the combination of mAb AC10364 with chemotherapeutic drugs demonstrated enhanced growth inhibitory effects on carcinoma cells. These results suggest that mAb AC10364 is a promising candidate for cancer therapy.
Asian Pacific journal of cancer prevention: APJCP 01/2014; 15(11):4423-8. · 1.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Environmental Determinants of Diabetes in the Young (TEDDY) planned biomarker discovery studies on longitudinal samples for persistent confirmed islet cell autoantibodies and type 1 diabetes (T1D) using dietary biomarkers, metabolomics, microbiome/viral metagenomics and gene expression.
This paper describes the details of planning the TEDDY biomarker discovery studies using a nested case-control design that was chosen as an alternative to the full cohort analysis. In the frame of a nested case-control design, it guides the choice of matching factors, selection of controls, preparation of external quality control samples, and reduction of batch effects along with proper sample allocation.
Our design is to reduce potential bias and retain study power while reduce the costs by limiting the numbers of samples requiring laboratory analyses. It also covers two primary end points (the occurrence of diabetes-related autoantibodies and the diagnosis of T1D). The resulting list of case-control matched samples for each laboratory was augmented with external quality control (QC) samples. This article is protected by copyright. All rights reserved.
Diabetes/Metabolism Research and Reviews 12/2013; · 2.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE
Our previous gene expression microarray studies identified a number of genes differentially expressed in patients with type 1 diabetes (T1D) and islet autoantibody-positive subjects. This study was designed to validate these gene expression changes in T1D patients and to identify gene expression changes in diabetes complications.RESEARCH DESIGH AND METHODS
We performed high-throughput real-time RT-PCR to validate gene expression changes in peripheral blood mononuclear cells (PBMCs) from a large sample set of 928 T1D patients and 922 control subjects.RESULTSOf the 18 genes analyzed here, eight genes (S100A8, S100A9, MNDA, SELL, TGFB1, PSMB3, CD74, and IL12A) had higher expression and three genes (GNLY, PSMA4, and SMAD7) had lower expression in T1D patients compared with control subjects, indicating that genes involved in inflammation, immune regulation, and antigen processing and presentation are significantly altered in PBMCs from T1D patients. Furthermore, one adhesion molecule (SELL) and three inflammatory genes mainly expressed by myeloid cells (S100A8, S100A9, and MNDA) were significantly higher in T1D patients with complications (odds ratio [OR] 1.3-2.6, adjusted P value = 0.005-10(-8)), especially those patients with neuropathy (OR 4.8-7.9, adjusted P value <0.005).CONCLUSIONS
These findings suggest that inflammatory mediators secreted mainly by myeloid cells are implicated in T1D and its complications.
[Show abstract][Hide abstract] ABSTRACT: Regulatory T cells (Treg), which play a pivotal role in maintaining immune homeostasis by suppressing the proliferation of effector T cells, have great therapeutic potential for autoimmune diseases and transplantation. However, progress on their clinical application has been hampered by the lack of high throughput screening (HTS) strategies for the systematic and rapid evaluation of existing drugs and the identification of novel drug candidates. In this report, we present an innovative in vitro HTS assay using CD4(+) T cells from Foxp3-GFP transgenic mice that specifically express the GFP signal in Foxp3(+) Treg cells detectable by FACS analysis in a high throughput manner. Systematic evaluation of 640 FDA-approved drugs revealed that 70 drugs increased the number of Treg cells with suppression function only in the presence of TGFβ, 75 drugs increased Treg numbers even in the absence of TGFβ, and 32 drugs increased Treg numbers synergistically with TGFβ. The identified Treg-promoting drugs include those previously known to induce Treg (rapamycin and retinoic acid), statins, glucocorticoids and drugs in many other categories. Furthermore, Treg cells cultured with the identified drugs possess surface and intracellular markers characteristic of natural Treg cells and possess suppressive function. These results suggest that this Treg HTS assay can be used to screen compound libraries to identify novel chemical entities for Treg-based immune therapies.
[Show abstract][Hide abstract] ABSTRACT: Mycophenolate mofetil (MMF), the prodrug of mycophenolic acid (MPA) which has been widely used for the prevention of acute graft rejection, is a potent inhibitor of inosine monophosphate dehydrogenase (IMPDH) that is up-regulated in many tumors and potentially a target for cancer therapy. MPA is known to inhibit cancer cell proliferation and induces apoptosis; however, the underlying molecular mechanisms remain elusive.
We first demonstrated MPA's antiproliferative and proapoptotic activities using in vitro studies of 13 cancer cell lines and a xenograft model. Key proteins involved in cell cycle, proliferation and apoptosis were analyzed by Western blotting.
In vitro treatment of thirteen cancer cell lines indicated that five cell lines (AGS, NCI-N87, HCT-8, A2780 and BxPC-3) are highly sensitive to MPA (IC50 < 0.5 μg/ml), four cell lines (Hs746T, PANC-1, HepG2 and MCF-7) are very resistant to MPA (IC50 > 20 μg/ml) and the four other cell lines (KATO III, SNU-1, K562 and HeLa) have intermediate sensitivity. The anticancer activity of MPA was confirmed in vivo using xenograft model with gastric AGS cell line. Further in vitro analyses using AGS cells indicated that MPA can potently induce cell cycle arrest and apoptosis as well as inhibition of cell proliferation. Targeted proteomic analyses indicate that many critical changes responsible for MPA's activities occur at the protein expression and phosphorylation levels. MPA-induced cell cycle arrest is achieved through reduction of many cell cycle regulators such as CDK4, BUB1, BOP1, Aurora A and FOXM1. We also demonstrate that MPA can inhibit the PI3K/AKT/mTOR pathway and can induce caspase-dependent apoptosis.
These results suggest that MPA has beneficial activities for anticancer therapy through diverse molecular pathways and biological processes.
International journal of clinical and experimental pathology 01/2013; 6(12):2880-6. · 2.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Activation of signal transducers and activators of transcription (STAT) proteins may be critical to their oncogenic functions as demonstrated by the development of B-cell lymphoma/leukemia in transgenic (TG) mice overexpressing a constitutively activated form of Stat5b. However, low incidence of CD8 T cell lymphoma was observed in B6 transgenic mice overexpressing a wild-type Stat5b (B6.Stat5b) despite of undetectable Stat5b phosphorylation and the rate of lymphomagenesis was markedly enhanced by immunization or the introduction of TCR transgenes . Here, we report that the wild-type Stat5b transgene leads to the acceleration and high incidence (74%) of CD8 T cell lymphoblastic lymphomas in the non-obese-diabetic (NOD) background. In contrast to the B6.Stat5b mice, Stat5b in transgenic NOD (NOD.Stat5b) mice is selectively and progressively phosphorylated in CD8 thymocytes. Stat5 phosphorylation also leads to up-regulation of many genes putatively relevant to tumorigenesis. Treatment of NOD.Stat5b mice with cancer chemopreventive agents Apigenin and Xanthohumol efficiently blocked lymphomagenesis through reduction of Stat5 phosphorylation and genes up-regulated in the NOD.Stat5b mice. These results suggest that NOD genetic background is critical to the Stat5b-mediated lymphomagenesis through regulation of Stat5 hyperactivation. NOD.Stat5b mouse is an excellent model for studying the molecular mechanisms underlying lymphomagenesis and testing novel chemoprevention strategies.
PLoS ONE 01/2013; 8(2):e56600. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inhibition of inosine monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA) can inhibit proliferation and induce apoptosis in cancer cells. This study investigated the underlying molecular mechanisms of MPA's anticancer activity.
A gastric cancer cell line (AGS) was treated with MPA and gene expression at different time points was analyzed using Illumina whole genome microarrays and selected genes were confirmed by real-time RT-PCR.
Transcriptomic profiling identified 1070 genes with ≥2 fold changes and 85 genes with >4 fold alterations. The most significantly altered biological processes by MPA treatment include cell cycle, apoptosis, cell proliferation and migration. MPA treatment altered at least ten KEGG pathways, of which eight (p53 signaling, cell cycle, pathways in cancer, PPAR signaling, bladder cancer, protein processing in ER, small cell lung cancer and MAPK signaling) are cancer-related. Among the earliest cellular events induced by MPA is cell cycle arrest which may be caused by six molecular pathways: 1) up-regulation of cyclins (CCND1 and CCNE2) and down-regulation of CCNA2 and CCNB1, 2) down-regulation of cyclin-dependent kinases (CDK4 and CDK5); 3) inhibition of cell division related genes (CDC20, CDC25B and CDC25C) and other cell cycle related genes (MCM2, CENPE and PSRC1), 4) activation of p53, which activates the cyclin-dependent kinase inhibitors (CDKN1A), 5) impaired spindle checkpoint function and chromosome segregation (BUB1, BUB1B, BOP1, AURKA, AURKB, and FOXM1); and 6) reduction of availability of deoxyribonucleotides and therefore DNA synthesis through down-regulation of the RRM1 enzyme. Cell cycle arrest is followed by inhibition of cell proliferation, which is mainly attributable to the inhibition of the PI3K/AKT/mTOR pathway, and caspase-dependent apoptosis due to up-regulation of the p53 and FAS pathways.
These results suggest that MPA has beneficial anticancer activity through diverse molecular pathways and biological processes.
American Journal of Translational Research 01/2013; 6(1):28-42.
[Show abstract][Hide abstract] ABSTRACT: Mycophenolic acid (MPA) is the metabolized product and active element of mycophenolate mofetil (MMF) that has been widely used for the prevention of acute graft rejection. MPA potently inhibits inosine monophosphate dehydrogenase (IMPDH) that is up-regulated in many tumors and MPA is known to inhibit cancer cell proliferation as well as fibroblast and endothelial cell migration. In this study, we demonstrated for the first time MPA's antimigratory and anti-invasion abilities of MPA-sensitive AGS (gastric cancer) cells. Genome-wide expression analyses using Illumina whole genome microarrays identified 50 genes with ≥2 fold changes and 15 genes with > 4 fold alterations and multiple molecular pathways implicated in cell migration. Real-time RT-PCR analyses of selected genes also confirmed the expression differences. Furthermore, targeted proteomic analyses identified several proteins altered by MPA treatment. Our results indicate that MPA modulates gastric cancer cell migration through down-regulation of a large number of genes (PRKCA, DOCK1, INF2, HSPA5, LRP8 and PDGFRA) and proteins (PRKCA, AKT, SRC, CD147 and MMP1) with promigratory functions as well as up-regulation of a number of genes with antimigratory functions (ATF3, SMAD3, CITED2 and CEAMCAM1). However, a few genes that may promote migration (CYR61 and NOS3) were up-regulated. Therefore, MPA's overall antimigratory role on cancer cells reflects a balance between promigratory and antimigratory signals influenced by MPA treatment.
PLoS ONE 01/2013; 8(11):e81702. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Biomarkers play critical roles in early detection, diagnosis and monitoring of therapeutic outcome and recurrence of cancer. Previous biomarker research on ovarian cancer (OC) has mostly focused on the discovery and validation of diagnostic biomarkers. The primary purpose of this study is to identify serum biomarkers for prognosis and therapeutic outcomes of ovarian cancer.
Forty serum proteins were analyzed in 70 serum samples from healthy controls (HC) and 101 serum samples from serous OC patients at three different disease phases: post diagnosis (PD), remission (RM) and recurrence (RC). The utility of serum proteins as OC biomarkers was evaluated using a variety of statistical methods including survival analysis.
Ten serum proteins (PDGF-AB/BB, PDGF-AA, CRP, sFas, CA125, SAA, sTNFRII, sIL-6R, IGFBP6 and MDC) have individually good area-under-the-curve (AUC) values (AUC = 0.69-0.86) and more than 10 three-marker combinations have excellent AUC values (0.91-0.93) in distinguishing active cancer samples (PD & RC) from HC. The mean serum protein levels for RM samples are usually intermediate between HC and OC patients with active cancer (PD & RC). Most importantly, five proteins (sICAM1, RANTES, sgp130, sTNFR-II and sVCAM1) measured at remission can classify, individually and in combination, serous OC patients into two subsets with significantly different overall survival (best HR = 17, p<10(-3)).
We identified five serum proteins which, when measured at remission, can accurately predict the overall survival of serous OC patients, suggesting that they may be useful for monitoring the therapeutic outcomes for ovarian cancer.
PLoS ONE 01/2013; 8(11):e78393. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The common genetic loci that independently influence the risk of type 1 diabetes have largely been determined. Their interactions with age-at-diagnosis of type 1 diabetes, sex, or the major susceptibility locus, HLA class II, remain mostly unexplored. A large collection of more than 14,866 type 1 diabetes samples (6,750 British diabetic individuals and 8,116 affected family samples of European descent) were genotyped at 38 confirmed type 1 diabetes-associated non-HLA regions and used to test for interaction of association with age-at-diagnosis, sex, and HLA class II genotypes using regression models. The alleles that confer susceptibility to type 1 diabetes at interleukin-2 (IL-2), IL2/4q27 (rs2069763) and renalase, FAD-dependent amine oxidase (RNLS)/10q23.31 (rs10509540), were associated with a lower age-at-diagnosis (P = 4.6 × 10(-6) and 2.5 × 10(-5), respectively). For both loci, individuals carrying the susceptible homozygous genotype were, on average, 7.2 months younger at diagnosis than those carrying the protective homozygous genotypes. In addition to protein tyrosine phosphatase nonreceptor type 22 (PTPN22), evidence of statistical interaction between HLA class II genotypes and rs3087243 at cytotoxic T-lymphocyte antigen 4 (CTLA4)/2q33.2 was obtained (P = 7.90 × 10(-5)). No evidence of differential risk by sex was obtained at any loci (P ≥ 0.01). Statistical interaction effects can be detected in type 1 diabetes although they provide a relatively small contribution to our understanding of the familial clustering of the disease.
[Show abstract][Hide abstract] ABSTRACT: The signal transducers and activators of transcription (STAT) family of proteins play a critical role in cytokine signaling required for fine tuning of immune regulation. Previous reports showed that a mutation (L327M) in the Stat5b protein leads to aberrant cytokine signaling in the NOD mice. To further elaborate the role of Stat5b in diabetes, we established a NOD transgenic mouse that over-expresses the wild type Stat5b gene. The incidences of spontaneous diabetes as well as cyclophosphamide-induced diabetes were significantly reduced and delayed in the Stat5b transgenic NOD mice compared to their littermate controls. The total cell numbers of CD4(+) T cells and especially CD8(+) T cells in the spleen and pancreatic lymph node were increased in the Stat5b transgenic NOD mice. Consistent with these findings, CD4(+) and CD8(+) T cells from the Stat5b transgenic NOD mice showed a higher proliferation capacity and up-regulation of multiple cytokines including IL-2, IFN-γ, TNF-α and IL-10 as well as anti-apoptotic gene Bcl-xl. Furthermore, the number and proportion of CD4(+)CD25(+) regulatory T cells were significantly increased in transgenic mice although in vitro suppression ability of the regulatory T-cells was not affected by the transgene. Our results suggest that Stat5b confers protection against diabetes in the NOD mice by regulating the numbers and function of multiple immune cell types, especially by up-regulating CD4(+)CD25(+) regulatory T cells.
Biochemical and Biophysical Research Communications 07/2012; 424(4):669-74. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Biomarkers are useful tools for research into type 1 diabetes (T1D) for a number of purposes, including elucidation of disease pathogenesis, risk prediction, and therapeutic monitoring. Susceptibility genes and islet autoantibodies are currently the most useful biomarkers for T1D risk prediction. However, these markers do not fully meet the needs of scientists and physicians for several reasons. First, improvement of the specificity and sensitivity is still desirable to achieve better positive predictive values. Second, autoantibodies appear relatively late in the disease process, thus limiting their value in early disease prediction. Third, the currently available biomarkers are not useful for assessing therapeutic outcomes because some are not involved in the disease process (autoantibodies) and others do not change during disease progression (susceptibility genes). Therefore, considerable effort has been devoted to the discovery of novel T1D biomarkers in the last three decades. The advent of high-throughput technologies for genetic, transcriptomic, and proteomic studies has allowed genome-wide examinations of genetic polymorphisms, global gene changes, and protein expression changes in T1D patients and prediabetic subjects. These large-scale studies resulted in the discovery of a large number of susceptibility genes and changes in gene and protein expression. While these studies have provided a number of novel biomarker candidates, their clinical benefits remain to be evaluated in prospective studies, and no new "star biomarker" has been identified until now. Previous studies suggest that significant improvements in study design and analytical methodologies have to be made to identify clinically relevant biomarkers. In this review, we discuss progress, opportunities, challenges, and future directions in the development of T1D biomarkers, mainly by focusing on the genetic, transcriptomic, and proteomic aspects.
The Review of Diabetic Studies 01/2012; 9(4):224-235.
[Show abstract][Hide abstract] ABSTRACT: Insulin-like growth factor binding proteins (IGFBPs) are associated with insulin resistance and accelerated micro- and macro-vascular complications of diabetes. We investigated the relationship between serum levels of IGFBP6 in type-1 diabetes (T1D) patients and diabetic complications. In this study, IGFBP6 was measured in the sera from 697 T1D patients and 681 healthy controls using a Luminex assay. Mean serum levels of IGFBP6 were higher in T1D patients than controls matched for sex and age (119.7 vs 130.6 ng/ml, p < 10(-4)). Subject age, sex and duration of disease have a significant impact on serum IGFBP6 levels in both T1D patients and healthy controls. Patients with complications have significantly higher mean serum IGFBP6 than patients without any complication (p = 3.5x10(-6)). More importantly, conditional logistic regression analysis suggested that T1D patients are more likely to have very high levels of serum IGFBP6 (in the 4(th) quartile) (OR = 1.7) than healthy controls. Furthermore, T1D patients with various complications are more likely to have very high levels of serum IGFBP6 (in the 4(th) quartile) than patients without any complication (OR = 1.7 - 22.9). These results indicate the clinical importance of measuring IGFBP6 to the better management of T1D patients.
International Journal of Clinical and Experimental Medicine 01/2012; 5(3):229-37. · 1.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Type 1 diabetes (T1D) is expected to cause significant changes in the serum proteome; however, few studies have systematically assessed the proteomic profile change associated with the disease. In this study, a semiquantitative spectral counting-based two dimensional liquid chromatography mass spectrometry platform was used to analyze serum samples from T1D patients and controls. In this discovery phase, significant differences were found for 21 serum proteins implicated in inflammation, oxidation, metabolic regulation, and autoimmunity. To assess the validity of these findings, six candidate proteins including adiponectin, insulin-like growth factor binding protein 2, serum amyloid protein A, C-reactive protein, myeloperoxidase, and transforming growth factor beta induced were selected for subsequent immune assays for 1139 T1D patients and 848 controls. A series of statistical analyses using cases and controls matched for age, sex, and genetic risk confirmed that T1D patients have significantly higher serum levels for four of the six proteins: adiponectin (odds ratio (OR) = 1.95, p = 10(-27)), insulin-like growth factor binding protein 2 (OR = 2.02, p < 10(-20)), C-reactive protein (OR = 1.13, p = 0.007), serum amyloid protein A (OR = 1.51, p < 10(-16)); whereas the serum levels were significantly lower in patients than controls for the two other proteins: transforming growth factor beta induced (OR = 0.74, p < 10(-5)) and myeloperoxidase (OR = 0.51, p < 10(-41)). Compared with subjects in the bottom quartile, subjects in the top quartile for adiponectin (OR = 6.29, p < 10(-37)), insulin-like growth factor binding protein 2 (OR = 7.95, p < 10(-46)), C-reactive protein (OR = 1.38, p = 0.025), serum amyloid protein A (OR = 3.36, p < 10(-16)) had the highest risk of T1D, whereas subjects in the top quartile of transforming growth factor beta induced (OR = 0.41, p < 10(-11)) and myeloperoxidase (OR = 0.10, p < 10(-43)) had the lowest risk of T1D. These findings provided valuable information on the proteomic changes in the sera of T1D patients.
[Show abstract][Hide abstract] ABSTRACT: Previous studies have noted a specific association between type 1 diabetes and insufficient levels of vitamin D, as well as polymorphisms within genes related to vitamin D pathways. Here, we examined whether serum levels or genotypes of the vitamin D-binding protein (VDBP), a molecule key to the biologic actions of vitamin D, specifically associate with the disorder.
A retrospective, cross-sectional analysis of VDBP levels used samples from 472 individuals of similar age and sex distribution, including 153 control subjects, 203 patients with type 1 diabetes, and 116 first-degree relatives of type 1 diabetic patients. Single nucleotide polymorphism (SNP) typing for VDBP polymorphisms (SNP rs4588 and rs7041) was performed on this cohort to determine potential genetic correlations. In addition, SNP analysis of a second sample set of banked DNA samples from 1,502 type 1 diabetic patients and 1,880 control subjects also was used to determine genotype frequencies.
Serum VDBP levels were highest in healthy control subjects (median 423.5 µg/mL [range 193.5-4,345.0; interquartile range 354.1-]586), intermediate in first-degree relatives (402.9 µg/mL [204.7-4,850.0; 329.6-492.4]), and lowest in type 1 diabetic patients (385.3 µg/mL [99.3-1,305.0; 328.3-473.0]; P = 0.003 vs. control subjects). VDBP levels did not associate with serum vitamin D levels, age, or disease duration. However, VDBP levels were, overall, lower in male subjects (374.7 µg/mL [188.9-1,602.0; 326.9-449.9]) than female subjects (433.4 µg/mL [99.3-4,850.0; 359.4-567.8]; P < 0.0001). It is noteworthy that no differences in genotype frequencies of the VDBP polymorphisms were associated with serum VDBP levels or between type 1 diabetic patients and control subjects.
Serum VDBP levels are decreased in those with type 1 diabetes. These studies suggest that multiple components in the metabolic pathway of vitamin D may be altered in type 1 diabetes and, collectively, have the potential to influence disease pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: The validation of putative biomarker candidates has become the major bottle-neck in protein biomarker development. Conventional immunoaffinity methods are limited by the availability of antibodies and kits. Here we demonstrate the feasibility of using selected reaction monitoring (SRM) without isotope labeling to achieve fast and reproducible quantification of serum proteins. The SRM/MRM assays for three standard serum proteins, including ceruloplasmin (CP), serum aymloid A (SAA) and sex hormone binding globulin (SHBG), have good linear ranges, generally 10(3) to 10(4) . There are almost perfect correlations between SRM intensities and the loaded peptide amounts (R(2) is usually ~0.99). Our data suggest that SRM/MRM is able to quantify proteins within the range of 0.2-2 fmol, which is comparable to the commercial ELISA/LUMINEX kits for these proteins. Excellent correlations between SRM/MRM and ELISA/LUMINEX assays were observed for SAA and SHBG (R(2)=0.928 and 0.851, respectively). However, the correlation between SRM/MRM and ELISA for CP is less desirable (R(2)=0.565). The reproducibility for SRM/MRM assays is generally very good but may depend on the proteins/peptides being analyzed (R(2)=0.931 and 0.882 for SAA and SHBG, and 0.723 for CP). The SRM/MRM assay without isotope labeling is a rapid and useful method for protein biomarker validation in a modest number of samples and is especially useful when other assays such as ELISA or LUMINEX are not available.
Rapid Communications in Mass Spectrometry 06/2011; 25(11):1583-8. · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite considerable efforts, the molecular and cellular events in lacrimal gland tissues initiating inflammatory responses leading to keratoconjunctivitis sicca (KCS), autoimmunity, and Sjögren's syndrome (SjS) have yet to be defined. To determine whether altered glandular homeostasis occurs before the onset of autoimmunity, a temporal transcriptome study was carried out in an animal model of primary SjS.
Using oligonucleotide microarrays, gene expression profiles were generated for lacrimal glands of C57BL/6.NOD-Aec1Aec2 mice 4 to 20 weeks of age. Pairwise analyses identified genes differentially expressed, relative to their 4-week expression, during the development of SjS-like disease. Statistical analyses defined differentially and coordinately expressed gene sets. The PANTHER (Protein ANalysis THrough Evolutionary Relationships) classification system was used to define annotated biological processes or functions.
Temporal transcript expression profiles of C57BL/6.NOD-Aec1Aec2 lacrimal glands before, or concomitant with, the first appearance of inflammatory cells revealed a highly restricted subset of differentially expressed genes encoding interactive extracellular matrix proteins, fibronectin, integrins, and syndecans. In contrast, genes encoding interepithelial junctional complex proteins defined alterations in tight junctions (TJ), adherens, desmosomes, and gap junctions, suggesting perturbations in the permeability of the paracellular spaces between epithelial barriers. Correlating with this were gene sets defining focal adhesion (FA) maturation and Ras/Raf-Mek/Erk signal transduction. Immunohistochemically, FAs were associated with infiltrating leukocytes and not with lacrimal epithelial cells.
For the first time, FA maturations are implicated as initial biomarkers of impending autoimmunity in lacrimal glands of SjS-prone mice. Changes in TJ complex genes support an increased movement of cells through paracellular spaces.