Identification of novel biomarkers for the prediction of subclinical coronary artery atherosclerosis in patients with rheumatoid arthritis: an exploratory analysis.

BACKGROUND: Cardiovascular (CV) risk estimation calculators for the general population underperform in patients with rheumatoid arthritis (RA). The purpose of this study was to identify relevant protein biomarkers that could be added to traditional CV risk calculators to improve the capacity of coronary artery calcification (CAC) prediction in individuals with RA. In a second step, we quantify the improvement of this prediction of CAC when these circulating biomarkers are added to standard risk scores. METHODS: A panel of 141 serum and plasma proteins, which represent a broad base of both CV and RA biology, were evaluated and prioritized as candidate biomarkers. Of these, 39 proteins were selected and measured by commercial ELISA or quantitative mass spectroscopy in 561 individuals with RA in whom a measure of CAC and frozen sera were available. The patients were randomly split 50:50 into a training/validation cohort. Discrimination (using area under the receiver operator characteristic curves) and re-classification (through net reclassification improvement and integrated discrimination improvement calculation) analyses were performed first in the training cohort and replicated in the validation cohort, to estimate the increase in prediction accuracy for CAC using the ACA/AHA (American College of Cardiology and the American Heart Association) score with, compared to without, addition of these circulating biomarkers. RESULTS: The model containing ACC/AHA score plus cytokines (osteopontin, cartilage glycoprotein-39, cystatin C, and chemokine (C-C motif) ligand 18) and plus quantitative mass spectroscopy biomarkers (serpin D1, paraoxonase, and clusterin) had a statistically significant positive net reclassifications index and integrated discrimination improvement for the prediction of CAC, using ACC/AHA score without any biomarkers as the reference category. These results were confirmed in the validation cohort. CONCLUSION: In this exploratory analysis, the addition of several circulating CV and RA biomarkers to a standard CV risk calculator yielded significant improvements in discrimination and reclassification for the presence of CAC in individuals with RA.

Validation of a novel multibiomarker test to assess rheumatoid arthritis disease activity.

OBJECTIVE: Quantitative assessment of disease activity in rheumatoid arthritis (RA) is important for patient management, and additional objective information may aid rheumatologists in clinical decision making. We validated a recently developed multibiomarker disease activity (MBDA) test relative to clinical disease activity in diverse RA cohorts. METHODS: Serum samples were obtained from the Index for Rheumatoid Arthritis Measurement, Brigham and Women's Hospital Rheumatoid Arthritis Sequential Study, and Leiden Early Arthritis Clinic cohorts. Levels of 12 biomarkers were measured and combined according to a prespecified algorithm to generate the composite MBDA score. The relationship of the MBDA score to clinical disease activity was characterized separately in seropositive and seronegative patients using Pearson's correlations and the area under the receiver operating characteristic curve (AUROC) to discriminate between patients with low and moderate/high disease activity. Associations between changes in MBDA score and clinical responses 6-12 weeks after initiation of anti-tumor necrosis factor or methotrexate treatment were evaluated by the AUROC. RESULTS: The MBDA score was significantly associated with the Disease Activity Score in 28 joints using the C-reactive protein level (DAS28-CRP) in both seropositive (AUROC 0.77, P < 0.001) and seronegative (AUROC 0.70, P < 0.001) patients. In subgroups based on age, sex, body mass index, and treatment, the MBDA score was associated with the DAS28-CRP (P < 0.05) in all seropositive and most seronegative subgroups. Changes in the MBDA score at 6-12 weeks could discriminate both American College of Rheumatology criteria for 50% improvement responses (P = 0.03) and DAS28-CRP improvement (P = 0.002). Changes in the MBDA score at 2 weeks were also associated with subsequent DAS28-CRP response (P = 0.02). CONCLUSION: Our findings establish the criterion and discriminant validity of a novel multibiomarker test as an objective measure of RA disease activity to aid in the management of RA in patients with this condition.

External Qi of Yan Xin Qigong induces cell death and gene expression alterations promoting apoptosis and inhibiting proliferation, migration and glucose metabolism in small-cell lung cancer cells.

Small-cell lung cancer (SCLC) is a highly malignant carcinoma with poor long-term survival. Effective treatment remains highly demanded. In the present study, we demonstrated that External Qi of Yan Xin Qigong (YXQ-EQ) exerted potent cytotoxic effect towards SCLC cell line NCI-H82 via induction of apoptosis. Global gene expression profiling identified 39 genes whose expression was altered by YXQ-EQ in NCI-82 cells. Among them, semi-quantitative RT-PCR and real-time qPCR analyses confirmed that the gene expression levels of apoptotic proteins death-associated protein kinase 2 and cell death-inducing DFFA-like effector b were upregulated, whereas that of oncoproteins DEK and MYCL1, cell migration-promoting proteins CD24 and integrin-alpha 9, and glycolytic enzyme aldolase A were downregulated. These findings suggest that YXQ-EQ may exert anticancer effect through modulating gene expression in a way that facilitates cancer cell apoptosis while represses proliferation, metastasis, and glucose metabolism.

Anthrax lethal toxin-induced gene expression changes in mouse lung.

A major virulence factor of Bacillus anthracis is the anthrax Lethal Toxin (LeTx), a bipartite toxin composed of Protective Antigen and Lethal Factor. Systemic administration of LeTx to laboratory animals leads to death associated with vascular leakage and pulmonary edema. In this study, we investigated whether systemic exposure of mice to LeTx would induce gene expression changes associated with vascular/capillary leakage in lung tissue. We observed enhanced susceptibility of A/J mice to death by systemic LeTx administration compared to the C57BL/6 strain. LeTx-induced groups of both up- and down-regulated genes were observed in mouse lungs 6 h after systemic administration of wild type toxin compared to lungs of mice exposed to an inactive mutant form of the toxin. Lungs of the less susceptible C57BL/6 strain showed 80% fewer differentially expressed genes compared to lungs of the more sensitive A/J strain. Expression of genes known to regulate vascular permeability was modulated by LeTx in the lungs of the more susceptible A/J strain. Unexpectedly, the largest set of genes with altered expression was immune specific, characterized by the up-regulation of lymphoid genes and the down-regulation of myeloid genes. Transcripts encoding neutrophil chemoattractants, modulators of tumor regulation and angiogenesis were also differentially expressed in both mouse strains. These studies provide new directions for the investigation of vascular leakage and pulmonary edema induced by anthrax LeTx.

Identification of outcome-correlated cytokine clusters in chronic lymphocytic leukemia.

Individual cytokines and groups of cytokines that might represent networks in chronic lymphocytic leukemia (CLL) were analyzed and their prognostic values determined. Serum levels of 23 cytokines were measured in 84 patients and 49 age-matched controls; 17 levels were significantly elevated in patients. Unsupervised hierarchical bicluster analysis identified 3 clusters (CLs) of highly correlated but differentially expressed cytokines: CL1 (CXCL9, CXCL10, CXCL11, CCL3, CCL4, CCL19, IL-5, IL-12, and IFNγ), CL2 (TNFα, IL-6, IL-8, and GM-CSF), and CL3 (IL-1ß, IL-2, IL-4, IL-15, IL-17, and IFNα). Combination scores integrating expression of CL1/CL2 or CL1/CL3 strongly correlated (P < .005) with time-to-first-treatment and overall survival (OS), respectively. Patients with the worst course had high CL1 and low CL2 or CL3 levels. Multivariate analysis revealed that CL1/CL2 combination score and immunoglobulin heavy chain variable region mutation status were independent prognostic indicators for time-to-first-treatment, whereas CL1/CL3 combination score and immunoglobulin heavy chain variable region mutation status were independent markers for OS. Thus, we identified groups of cytokines differentially expressed in CLL that are independent prognostic indicators of aggressive disease and OS. These findings indicate the value of multicytokine analyses for prognosis and suggest therapeutic strategies in CLL aimed at reducing CL1 and increasing CL2/CL3 cytokines.

Internal standard-based analysis of microarray data2--analysis of functional associations between HVE-genes.

In this work we apply the Internal Standard-based analytical approach that we described in an earlier communication and here we demonstrate experimental results on functional associations among the hypervariably-expressed genes (HVE-genes). Our working assumption was that those genetic components, which initiate the disease, involve HVE-genes for which the level of expression is undistinguishable among healthy individuals and individuals with pathology. We show that analysis of the functional associations of the HVE-genes is indeed suitable to revealing disease-specific differences. We show also that another possible exploit of HVE-genes for characterization of pathological alterations is by using multivariate classification methods. This in turn offers important clues on naturally occurring dynamic processes in the organism and is further used for dynamic discrimination of groups of compared samples. We conclude that our approach can uncover principally new collective differences that cannot be discerned by individual gene analysis.

Gene network analysis of bone marrow mononuclear cells reveals activation of multiple kinase pathways in human systemic lupus erythematosus.

BACKGROUND: Gene profiling studies provide important information for key molecules relevant to a disease but are less informative of protein-protein interactions, post-translational modifications and regulation by targeted subcellular localization. Integration of genomic data and construction of functional gene networks may provide additional insights into complex diseases such as systemic lupus erythematosus (SLE). METHODOLOGY/PRINCIPAL FINDINGS: We analyzed gene expression microarray data of bone marrow mononuclear cells (BMMCs) from 20 SLE patients (11 with active disease) and 10 controls. Gene networks were constructed using the bioinformatic tool Ingenuity Gene Network Analysis. In SLE patients, comparative analysis of BMMCs genes revealed a network with 19 central nodes as major gene regulators including ERK, JNK, and p38 MAP kinases, insulin, Ca(2+) and STAT3. Comparison between active versus inactive SLE identified 30 central nodes associated with immune response, protein synthesis, and post-transcriptional modification. A high degree of identity between networks in active SLE and non-Hodgkin's lymphoma (NHL) patients was found, with overlapping central nodes including kinases (MAPK, ERK, JNK, PKC), transcription factors (NF-kappaB, STAT3), and insulin. In validation studies, western blot analysis in splenic B cells from 5-month-old NZB/NZW F1 lupus mice showed activation of STAT3, ITGB2, HSPB1, ERK, JNK, p38, and p32 kinases, and downregulation of FOXO3 and VDR compared to normal C57Bl/6 mice. CONCLUSIONS/SIGNIFICANCE: Gene network analysis of lupus BMMCs identified central gene regulators implicated in disease pathogenesis which could represent targets of novel therapies in human SLE. The high similarity between active SLE and NHL networks provides a molecular basis for the reported association of the former with lymphoid malignancies.

Idiopathic inflammatory myopathies, signified by distinctive peripheral cytokines, chemokines and the TNF family members B-cell activating factor and a proliferation inducing ligand.

OBJECTIVE: Serum cytokines play an important role in the pathogenesis of myositis by initiating and perpetuating various cellular and humoral autoimmune processes. The aim of the present study was to describe a broad spectrum of T- and B-cell cytokines, growth factors and chemokines in patients with idiopathic inflammatory myopathies (IIMs) and healthy individuals. METHODS: A protein array system, denoted as multiplex cytokine assay was utilized to measure simultaneously the levels of 24 circulating cytokines, including B-cell activating factor (BAFF) and a proliferation inducing ligand (APRIL) of patients with IIMs and healthy individuals. Additionally, correlational clustering and discriminant function analysis (DFA), two multivariate, supervised analysis methods were employed to identify a subset of biomarkers in order to describe potential functional interrelationships among these pathological cytokines. RESULTS: Univariate analysis demonstrated that a complex set of immune and inflammatory modulating cytokines are significantly up-regulated in patients with IIMs relative to unaffected controls including IL-10, IL-13, IFN-α, epidermal growth factor (EGF), VEGF, fibroblast growth factor (FGF), CCL3 [macrophage inflammatory protein (MIP-1α)], CCL4 (MIP-1ß) and CCL11 (eotaxin), whereas G-CSF was significantly reduced in IIM patients. Correlational clustering was able to discriminate between, and hence sub-classify patients with IIMs. DFA identified EGF, IFN-α, VEGF, CCL3 (MIP-1α) and IL-12p40, as analytes with the strongest discriminatory power among various myositis patients and controls. CONCLUSIONS: Our findings suggest that these factors modulate myositis pathology and help to identify differences between subsets of the disease.

Scaffold-guided subchondral bone repair: implication of neutrophils and alternatively activated arginase-1+ macrophages.

BACKGROUND: Microfracture and drilling elicit a cartilage repair whose quality depends on subchondral bone repair. Alternatively activated (AA) macrophages express arginase-1, release angiogenic factors, and could be potential mediators of trabecular bone repair. HYPOTHESIS: Chitosan-glycerol phosphate (GP)/blood implants elicit arginase-1+ macrophages in vivo through neutrophil-dependent mechanisms and improve trabecular bone repair of drilled defects compared with drilling alone. STUDY DESIGN: Controlled laboratory study. METHODS: Bilateral trochlear cartilage defects were created in 15 rabbits, microdrilled, and treated or not with chitosan-GP/blood implant to analyze AA macrophages, CD-31+ blood vessels, bone, and cartilage repair after 1, 2, or 8 weeks. Neutrophil and macrophage chemotaxis to rabbit subcutaneous implants of autologous blood and chitosan-GP (+/-blood) was quantified at 1 or 7 days. In vitro, sera from human chitosan-GP/blood and whole blood clots cultured at 37 degrees C were analyzed by proteomics and neutrophil chemotaxis assays. RESULTS: Chitosan-GP/blood clots and whole blood clots released a similar profile of chemotactic factors (PDGF-BB, IL-8/CXCL8, MCP-1/CCL2, and no IL-1beta or IL-6), although chitosan clot sera attracted more neutrophils in vitro. Subcutaneous chitosan-GP (+/-blood) implants attracted more neutrophils (P < .001) and AA macrophages than whole blood clots in vivo. In repairing subchondral drill holes, chitosan-GP/blood implant attracted more AA macrophages at 1 and 2 weeks and more blood vessels at 2 weeks compared with drilled controls. Treatment elicited a more complete woven bone repair at 8 weeks than controls (P = .0011) with a more uniform, integrated collagen type II+ cartilage repair tissue. CONCLUSION AND CLINICAL RELEVANCE: AA macrophages may play a role in the regeneration of subchondral bone, and chitosan-GP can attract and transiently accumulate these cells in the repair tissue. The resulting improved subchondral repair could be advantageous toward enhancing integration of a restored chondral surface to the subchondral bone.

Clcn5 knockout mice exhibit novel immunomodulatory effects and are more susceptible to dextran sulfate sodium-induced colitis.

Although the intracellular Cl(-)/H(+) exchanger Clc-5 is expressed in apical intestinal endocytic compartments, its pathophysiological role in the gastrointestinal tract is unknown. In light of recent findings that CLC-5 is downregulated in active ulcerative colitis (UC), we tested the hypothesis that loss of CLC-5 modulates the immune response, thereby inducing susceptibility to UC. Acute dextran sulfate sodium (DSS) colitis was induced in Clcn5 knockout (KO) and wild-type (WT) mice. Colitis, monitored by disease activity index, histological activity index, and myeloperoxidase activity were significantly elevated in DSS-induced Clcn5 KO mice compared with those in WT mice. Comprehensive serum multiplex cytokine profiling demonstrated a heightened Th1-Th17 profile (increased TNF-alpha, IL-6, and IL-17) in DSS-induced Clcn5 KO mice compared with that in WT DSS colitis mice. Interestingly, Clcn5 KO mice maintained on a high vitamin D diet attenuated DSS-induced colitis. Immunofluorescence and Western blot analyses of colonic mucosa validated the systemic cytokine patterns and further revealed enhanced activation of the NF-kappaB pathway in DSS-induced Clcn5 KO mice compared with those in WT mice. Intriguingly, high baseline levels of IL-6 and phospho-IkappaB were observed in Clcn5 KO mice, suggesting a novel immunopathogenic role for the functional defects that result from the loss of Clc-5. Our studies demonstrate that the loss of Clc-5 1) exhibits IL-6-mediated immunopathogenesis, 2) significantly exacerbated DSS-induced colitis, which is influenced by dietary factors, including vitamin D, and 3) portrays distinct NF-kappaB-modulated Th1-Th17 immune dysregulation, implying a role for CLC-5 in the immunopathogenesis of UC.

Estrogen receptor signaling promotes dendritic cell differentiation by increasing expression of the transcription factor IRF4.

During inflammation, elevated granulocyte macrophage-colony-stimulating factor (GM-CSF) directs the development of new dendritic cells (DCs). This pathway is influenced by environmental factors, and we previously showed that physiologic levels of estradiol, acting through estrogen receptor alpha (ERalpha), promote the GM-CSF-mediated differentiation of a CD11b(+) DC subset from myeloid progenitors (MPs). We now have identified interferon regulatory factor 4 (IRF4), a transcription factor induced by GM-CSF and critical for CD11b(+) DC development in vivo, as a target of ERalpha signaling during this process. In MPs, ERalpha potentiates and sustains GM-CSF induction of IRF4. Furthermore, retroviral delivery of the Irf4 cDNA to undifferentiated ERalpha(-/-) bone marrow cells restored the development of the estradiol/ERalpha-dependent DC population, indicating that an elevated amount of IRF4 protein substitutes for ERalpha signaling. Thus at an early stage in the MP response to GM-CSF, ERalpha signaling induces an elevated amount of IRF4, which leads to a developmental program underlying CD11b(+) DC differentiation.

Identification of RGS1 as a candidate biomarker for undifferentiated spondylarthritis by genome-wide expression profiling and real-time polymerase chain reaction.

OBJECTIVE: To compare gene expression profiles between ankylosing spondylitis (AS) and undifferentiated spondylarthritis (uSpA) patients with inflammatory low back pain. METHODS: Peripheral blood mononuclear cells (PBMCs) from patients with AS, patients with uSpA, and healthy subjects were screened using genome-wide microarrays, followed by validation by real-time polymerase chain reaction (PCR). RESULTS: Microarray profiling and real-time PCR assays showed only minor differences between AS patients and healthy subjects. In contrast, 20 genes were strikingly more highly expressed in uSpA patients. Regulator of G protein signaling 1 (RGS1) was identified as the most useful biomarker for distinguishing uSpA patients, and to a lesser extent AS patients, from control subjects (P = 2.3 x 10(-7) and 6.7 x 10(-3), respectively). These findings were verified in an independent cohort that also included patients with rheumatoid arthritis and patients with mechanical low back pain. The receiver operating characteristic area under the curve values in the first and second cohorts of uSpA patients were 0.99 and 0.93, respectively (P = 1 x 10(-4)). To evaluate the possible derivation of RGS1, we cultured a monocyte-derived cell line with a panel of cytokines and chemokines. RGS1 was significantly induced either by tumor necrosis factor alpha (TNFalpha) or by interleukin-17 (IL-17). CONCLUSION: Our findings indicate that uSpA PBMCs carry strikingly more highly expressed genes compared with PBMCs from AS patients or healthy subjects, and that TNFalpha- and IL-17-inducible RGS1 is a potential biomarker for uSpA, and to a lesser extent for AS, with inflammatory low back pain.

Effect of chitosan particles and dexamethasone on human bone marrow stromal cell osteogenesis and angiogenic factor secretion.

Chitosan is a polysaccharide scaffold used to enhance cartilage repair during treatments involving bone marrow stimulation, and it is reported to increase angiogenesis and osteogenesis in vivo. Here, we tested the hypotheses that addition of chitosan particles to the media of human bone marrow stromal cell (BMSC) cultures stimulates osteogenesis by promoting osteoblastic differentiation and by favoring the release of angiogenic factors in vitro. Confluent BMSCs were cultured for 3 weeks with 16% fetal bovine serum, ascorbate-2-phosphate and disodium beta-glycerol phosphate, in the absence or presence of dexamethasone, an anti-inflammatory glucocorticoid commonly used as an inducer of BMSC osteoblast differentiation in vitro. As expected, dexamethasone slowed cell division, stimulated alkaline phosphatase activity and enhanced matrix mineralization. Added chitosan particles accumulated intra- and extracellularly and, while not affecting most osteogenic features, they inhibited osteocalcin release to the media at day 14 and interfered with mineralized matrix deposition. Interestingly, dexamethasone promoted cell attachment and suppressed the release and activation of matrix metalloprotease-2 (MMP-2). While chitosan particles had no effect on the release of angiogenic factors, dexamethasone significantly inhibited (p<0.05 to p<0.0001) the release of vascular endothelial growth factor (VEGF), granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), interleukins 1beta, 4, 6, and 10 (IL-1beta, IL-4, IL-6, IL-10), and a host of other inflammatory factors that were constitutively secreted by BMSCs. These results demonstrate that chitosan particles alone are not sufficient to promote osteoblast differentiation of BMSCs in vitro, and suggest that chitosan promotes osteogenesis in vivo through indirect mechanisms. Our data further show that continuous addition of dexamethasone promotes osteoblastic differentiation in vitro partly by inhibiting gelatinase activity and by suppressing inflammatory cytokines which result in increased cell attachment and cell cycle exit.

Gene expression in systemic lupus erythematosus: bone marrow analysis differentiates active from inactive disease and reveals apoptosis and granulopoiesis signatures.

OBJECTIVE: The cells of the immune system originate from the bone marrow, where many of them also mature. This study was undertaken to examine gene expression in the bone marrow of patients with systemic lupus erythematosus (SLE), in order to better understand the aberrant immune response in this disease. METHODS: Bone marrow mononuclear cells (BMMCs) from 20 SLE patients (11 with active disease and 9 with inactive disease) and peripheral blood mononuclear cells (PBMCs) from 27 patients (16 with active disease and 11 with inactive disease) were studied; BMMCs and PBMCs from 7 healthy individuals and 3 osteoarthritis patients were studied as controls. Samples were analyzed on genome-scale DNA microarrays, with 21,329 genes represented. RESULTS: We identified 102 genes involved in various biologic processes that were differentially expressed between patient and control BMMCs; 53 of them are genes that are involved in major networks, including cell death, growth, signaling, and proliferation. Comparative analysis revealed 88 genes that were differentially expressed between bone marrow and blood, the majority of which are involved in cell growth and differentiation, cellular movement and morphology, immune response, and other hematopoietic cell functions. Unsupervised clustering of highly expressed genes revealed 2 major SLE patient clusters (active disease and inactive disease) based on gene expression in bone marrow, but not in peripheral blood. The up-regulated genes in the bone marrow of patients with active disease included genes involved in cell death and granulopoiesis. CONCLUSION: Microarray analysis of the bone marrow differentiated active from inactive SLE and provided further evidence of the role of apoptosis and granulocytes in the pathogenesis of the disease.

From microarray to biology: an integrated experimental, statistical and in silico analysis of how the extracellular matrix modulates the phenotype of cancer cells.

A statistically robust and biologically-based approach for analysis of microarray data is described that integrates independent biological knowledge and data with a global F-test for finding genes of interest that minimizes the need for replicates when used for hypothesis generation. First, each microarray is normalized to its noise level around zero. The microarray dataset is then globally adjusted by robust linear regression. Second, genes of interest that capture significant responses to experimental conditions are selected by finding those that express significantly higher variance than those expressing only technical variability. Clustering expression data and identifying expression-independent properties of genes of interest including upstream transcriptional regulatory elements (TREs), ontologies and networks or pathways organizes the data into a biologically meaningful system. We demonstrate that when the number of genes of interest is inconveniently large, identifying a subset of "beacon genes" representing the largest changes will identify pathways or networks altered by biological manipulation. The entire dataset is then used to complete the picture outlined by the "beacon genes." This allow construction of a structured model of a system that can generate biologically testable hypotheses. We illustrate this approach by comparing cells cultured on plastic or an extracellular matrix which organizes a dataset of over 2,000 genes of interest from a genome wide scan of transcription. The resulting model was confirmed by comparing the predicted pattern of TREs with experimental determination of active transcription factors.

Systems biology approach for mapping the response of human urothelial cells to infection by Enterococcus faecalis.

BACKGROUND: To better understand the response of urinary epithelial (urothelial) cells to Enterococcus faecalis, a uropathogen that exhibits resistance to multiple antibiotics, a genome-wide scan of gene expression was obtained as a time series from urothelial cells growing as a layered 3-dimensional culture similar to normal urothelium. We herein describe a novel means of analysis that is based on deconvolution of gene variability into technical and biological components. RESULTS: Analysis of the expression of 21,521 genes from 30 minutes to 10 hours post infection, showed 9553 genes were expressed 3 standard deviations (SD) above the system zero-point noise in at least 1 time point. The asymmetric distribution of relative variances of the expressed genes was deconvoluted into technical variation (with a 6.5% relative SD) and biological variation components (>3 SD above the mode technical variability). These 1409 hypervariable (HV) genes encapsulated the effect of infection on gene expression. Pathway analysis of the HV genes revealed an orchestrated response to infection in which early events included initiation of immune response, cytoskeletal rearrangement and cell signaling followed at the end by apoptosis and shutting down cell metabolism. The number of poorly annotated genes in the earliest time points suggests heretofore unknown processes likely also are involved. CONCLUSION: Enterococcus infection produced an orchestrated response by the host cells involving several pathways and transcription factors that potentially drive these pathways. The early time points potentially identify novel targets for enhancing the host response. These approaches combine rigorous statistical principles with a biological context and are readily applied by biologists.

Discriminators of mouse bladder response to intravesical Bacillus Calmette-Guerin (BCG).

BACKGROUND: Intravesical Bacillus Calmette-Guerin (BCG) is an effective treatment for bladder superficial carcinoma and it is being tested in interstitial cystitis patients, but its precise mechanism of action remains poorly understood. It is not clear whether BCG induces the release of a unique set of cytokines apart from its pro-inflammatory effects. Therefore, we quantified bladder inflammatory responses and alterations in urinary cytokine protein induced by intravesical BCG and compared the results to non-specific pro-inflammatory stimuli (LPS and TNF-alpha). We went further to determine whether BCG treatment alters cytokine gene expression in the urinary bladder. METHODS: C57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-alpha. Morphometric analyses were conducted in bladders isolated from all groups and urine was collected for multiplex analysis of 18 cytokines. In addition, chromatin immune precipitation combined with real-time polymerase chain reaction assay (CHIP/Q-PCR) was used to test whether intravesical BCG would alter bladder cytokine gene expression. RESULTS: Acute BCG instillation induced edema which was progressively replaced by an inflammatory infiltrate, composed primarily of neutrophils, in response to weekly administrations. Our morphological analysis suggests that these polymorphonuclear neutrophils are of prime importance for the bladder responses to BCG. Overall, the inflammation induced by BCG was higher than LPS or TNF-alpha treatment but the major difference observed was the unique granuloma formation in response to BCG. Among the cytokines measured, this study highlighted the importance of IL-1beta, IL-2, IL-3, IL-4, IL-6, IL-10, IL-17, GM-CSF, KC, and Rantes as discriminators between generalized inflammation and BCG-specific inflammatory responses. CHIP/Q-PCR indicates that acute BCG instillation induced an up-regulation of IL-17A, IL-17B, and IL-17RA, whereas chronic BCG induced IL-17B, IL-17RA, and IL-17RB. CONCLUSION: To the best of our knowledge, the present work is the first to report that BCG induces an increase in the IL-17 family genes. In addition, BCG induces a unique type of persisting bladder inflammation different from TNF-alpha, LPS, and, most likely, other classical pro-inflammatory stimuli.

The inflammatory and normal transcriptome of mouse bladder detrusor and mucosa.

BACKGROUND: An organ such as the bladder consists of complex, interacting set of tissues and cells. Inflammation has been implicated in every major disease of the bladder, including cancer, interstitial cystitis, and infection. However, scanty is the information about individual detrusor and urothelium transcriptomes in response to inflammation. Here, we used suppression subtractive hybridizations (SSH) to determine bladder tissue- and disease-specific genes and transcriptional regulatory elements (TRE)s. Unique TREs and genes were assembled into putative networks. RESULTS: It was found that the control bladder mucosa presented regulatory elements driving genes such as myosin light chain phosphatase and calponin 1 that influence the smooth muscle phenotype. In the control detrusor network the Pax-3 TRE was significantly over-represented. During development, the Pax-3 transcription factor (TF) maintains progenitor cells in an undifferentiated state whereas, during inflammation, Pax-3 was suppressed and genes involved in neuronal development (synapsin I) were up-regulated. Therefore, during inflammation, an increased maturation of neural progenitor cells in the muscle may underlie detrusor instability. NF-kappaB was specifically over-represented in the inflamed mucosa regulatory network. When the inflamed detrusor was compared to control, two major pathways were found, one encoding synapsin I, a neuron-specific phosphoprotein, and the other an important apoptotic protein, siva. In response to LPS-induced inflammation, the liver X receptor was over-represented in both mucosa and detrusor regulatory networks confirming a role for this nuclear receptor in LPS-induced gene expression. CONCLUSION: A new approach for understanding bladder muscle-urothelium interaction was developed by assembling SSH, real time PCR, and TRE analysis results into regulatory networks. Interestingly, some of the TREs and their downstream transcripts originally involved in organogenesis and oncogenesis were also activated during inflammation. The latter represents an additional link between inflammation and cancer. The regulatory networks represent key targets for development of novel drugs targeting bladder diseases.

Analysis of the interaction of extracellular matrix and phenotype of bladder cancer cells.

BACKGROUND: The extracellular matrix has a major effect upon the malignant properties of bladder cancer cells both in vitro in 3-dimensional culture and in vivo. Comparing gene expression of several bladder cancer cells lines grown under permissive and suppressive conditions in 3-dimensional growth on cancer-derived and normal-derived basement membrane gels respectively and on plastic in conventional tissue culture provides a model system for investigating the interaction of malignancy and extracellular matrix. Understanding how the extracellular matrix affects the phenotype of bladder cancer cells may provide important clues to identify new markers or targets for therapy. METHODS: Five bladder cancer cell lines and one immortalized, but non-tumorigenic, urothelial line were grown on Matrigel, a cancer-derived ECM, on SISgel, a normal-derived ECM, and on plastic, where the only ECM is derived from the cells themselves. The transcriptomes were analyzed on an array of 1186 well-annotated cancer derived cDNAs containing most of the major pathways for malignancy. Hypervariable genes expressing more variability across cell lines than a set expressing technical variability were analyzed further. Expression values were clustered, and to identify genes most likely to represent biological factors, statistically over-represented ontologies and transcriptional regulatory elements were identified. RESULTS: Approximately 400 of the 1186 total genes were expressed 2 SD above background. Approximately 100 genes were hypervariable in cells grown on each ECM, but the pattern was different in each case. A core of 20 were identified as hypervariable under all 3 growth conditions, and 33 were hypervariable on both SISgel and Matrigel, but not on plastic. Clustering of the hypervariable genes showed very different patterns for the same 6 cell types on the different ECM. Even when loss of cell cycle regulation was identified, different genes were involved, depending on the ECM. Under the most permissive conditions of growth where the malignant phenotype was fully expressed, activation of AKT was noted. TGFbeta1 signaling played a major role in the response of bladder cancer cells to ECM. Identification of TREs on genes that clustered together suggested some clustering was driven by specific transcription factors. CONCLUSION: The extracellular matrix on which cancer cells are grown has a major effect on gene expression. A core of 20 malignancy-related genes were not affected by matrix, and 33 were differentially expressed on 3-dimensional culture as opposed to plastic. Other than these genes, the patterns of expression were very different in cells grown on SISgel than on Matrigel or even plastic, supporting the hypothesis that growth of bladder cancer cells on normal matrix suppresses some malignant functions. Unique underlying regulatory networks were driving gene expression and could be identified by the approach outlined here.

17beta-estradiol (betaE2) protects human retinal Müller cell against oxidative stress in vitro: evaluation of its effects on gene expression by cDNA microarray.

17beta-estradiol (betaE(2)) is an effective neuroprotectant against hydrogen peroxide (H(2)O(2))-induced retinal neuronal cell death and light-induced photoreceptor degeneration. Müller cells are the principal macroglia responsible for supporting retinal neuronal survival, information processing and removing metabolic waste. However, the role of betaE(2) on human Müller cells is unclear. In this study, the effects of betaE(2) on human Müller cell survival and gene expression were examined. Our data revealed that betaE(2) is able to increase human Müller cell viability after exposure to H(2)O(2) through inhibition of apoptosis. Microarray analysis revealed significant changes in the expression of 69 genes (total of 21,324 genes screened) in cultured human Müller cells 6 h after betaE(2) treatment. Four of the betaE(2)-responsive genes [thrombospondin 1 (TSP1), mitogen-activated protein kinase kinase kinase 3 (MAP3K3), large conductance calcium-activated potassium channel beta2 subunit (KCNMB2), and SRY (sex-determining region Y)-box 11 (SOX11)] were validated by both real-time qRT-PCR and semi-quantitative RT-PCR. Interestingly, exposure of human Müller cells to betaE(2) increased pigment epithelium-derived factor (PEDF) gene expression as measured by both RT-PCR and real time qRT-PCR. Our data demonstrate, for the first time, that betaE(2) protects cultured human Müller cells against H(2)O(2)-induced cell death through the inhibition of apoptosis. This protective effect may operate through regulation of genes, such as TSP1, MAP3K3, SOX11, TSP1, and PEDF, and may in turn exert an important role in protecting retinal neurons.