A multi-biomarker score measures rheumatoid arthritis disease activity in the BeSt study.

OBJECTIVE: To evaluate a multi-biomarker disease activity (MBDA) score, a novel index based on 12 serum proteins, as a tool to guide management of RA patients. METHODS: A total of 125 patients with RA from the Behandel Strategieën study were studied. Clinical data and serum samples were available from 179 visits, 91 at baseline and 88 at year 1. In each serum sample, 12 biomarkers were measured by quantitative multiplex immunoassays and the concentrations were used as input to a pre-specified algorithm to calculate MBDA scores. RESULTS: MBDA scores had significant correlation with DAS28-ESR (Spearman's ρ = 0.66, P < 0.0001) and also correlated with simplified disease activity index, clinical disease activity index and HAQ Disability Index (all P < 0.0001). Changes in MBDA between baseline and year 1 were also correlated with changes in DAS28-ESR (ρ = 0.55, P < 0.0001). Groups stratified by European League Against Rheumatism disease activity (DAS28-ESR ≤ 3.2, 3.2-5.1 and > 5.1) had significantly different MBDA scores (P < 0.0001) and MBDA score could discriminate ACR/EULAR Boolean remission with an area under the receiver operating characteristic curve of 0.83 (P < 0.0001). CONCLUSION: The MBDA score reflects current clinical disease activity and can track changes in disease activity over time.

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.

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.

Performance of a multi-biomarker score measuring rheumatoid arthritis disease activity in the CAMERA tight control study.

OBJECTIVES: To evaluate the performance of individual biomarkers and a multi-biomarker disease activity (MBDA) score in the early rheumatoid arthritis (RA) patient population from the computer assisted management in early rheumatoid arthritis (CAMERA) study. METHODS: Twenty biomarkers were measured in the CAMERA cohort, in which patients were treated with either intensive or conventional methotrexate-based treatment strategies. The MBDA score was calculated using the concentrations of 12 biomarkers (SAA, IL-6, TNF-RI, VEGF-A, MMP-1, YKL-40, MMP-3, EGF, VCAM-1, leptin, resistin and CRP) according to a previously trained algorithm. The performance of the scores was evaluated relative to clinical disease activity assessments. Change in MBDA score over time was assessed by paired Wilcoxon rank sum test. Logistic regression was used to evaluate the ability of disease activity measures to predict radiographic progression. RESULTS: The MBDA score had a significant correlation with the disease activity score based on 28 joints-C reactive protein (DAS28-CRP) (r=0.72; p<0.001) and an area under the receiver operating characteristic curve for distinguishing remission/low from moderate/high disease activity of 0.86 (p<0.001) using a DAS28-CRP cut-off of 2.7. In multivariate analysis the MBDA score, but not CRP, was an independent predictor of disease activity measures. Additionally, mean (SD) MBDA score decreased from 53 (18) at baseline to 39 (16) at 6 months in response to study therapy (p<0.0001). Neither MBDA score nor clinical variables were predictive of radiographic progression. CONCLUSIONS: This multi-biomarker test performed well in the assessment of disease activity in RA patients in the CAMERA study. Upon further validation, this test could be used to complement currently available disease activity measures and improve patient care and outcomes.

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.

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.

Erroneous augmentation of multiplex assay measurements in patients with rheumatoid arthritis due to heterophilic binding by serum rheumatoid factor.

OBJECTIVE: Serum rheumatoid factor (RF) and other heterophilic antibodies potentially interfere with antibody-based immunoassays by nonspecifically binding detection reagents. The purpose of this study was to assess whether these factors confound multiplex-based immunoassays, which are used with increasing frequency to measure cytokine and chemokine analytes in patients with rheumatoid arthritis (RA). METHODS: We performed multiplex immunoassays using different platforms to measure analyte concentrations in RA patient samples. Samples were depleted of RF by column-based affinity absorption or were exposed to agents that block heterophilic binding activity. RESULTS: In RA patients with high-titer RF, 69% of analytes demonstrated at least a 2-fold stronger multiplex signal in non-RF-depleted samples as compared to RF-depleted samples. This degree of erroneous signal amplification was less frequent in low-titer RF samples (17% of analytes; P < 0.0000001). Signal amplification by heterophilic antibodies was blocked effectively by HeteroBlock (≥ 150 µg/ml). In 35 RA patients, multiplex signals for 14 of 22 analytes were amplified erroneously in unblocked samples as compared to blocked samples (some >100-fold), but only in patients with high-titer RF (P < 0.002). Two other blocking agents, heterophilic blocking reagent and immunoglobulin-inhibiting reagent, also blocked heterophilic activity. CONCLUSION: All multiplex protein detection platforms we tested exhibited significant confounding by RF or other heterophilic antibodies. These findings have broad-reaching implications in the acquisition and interpretation of data derived from multiplex immunoassay testing of RA patient serum and possibly also in other conditions in which RF or other heterophilic antibodies may be present. Several available blocking agents effectively suppressed this erroneous signal amplification in the multiplex platforms tested.

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.

COVID-19 Test Us: A Case for Embedding Ethics and Regulatory Expertise.

A key aspect of the National Institutes of Health (NIH) funded Rapid Acceleration of Diagnostics (RADx) Tech program was an active Clinical Studies Core including Committees with unique expertise to facilitate the development and implementation of studies to test novel diagnostic devices for Covid-19. The Ethics and Human Subjects Oversight Team (EHSO) was tasked to provide ethics and regulatory expertise to stakeholders in the RADx Tech effort. The EHSO developed a set of Ethical Principles to guide the overall effort and provided consultation on a wide range of ethical and regulatory concerns. Having access to a pool of experts with ethical and regulatory knowledge who met weekly to tackle issues of importance to the investigators was critical to the overall success of the project.

FOXP3 inhibits activation-induced NFAT2 expression in T cells thereby limiting effector cytokine expression.

The forkhead DNA-binding protein FOXP3 is critical for the development and suppressive function of CD4(+)CD25(+) regulatory T cells (T(REG)), which play a key role in maintaining self-tolerance. Functionally, FOXP3 is capable of repressing transcription of cytokine genes regulated by NFAT. Various mechanisms have been proposed by which FOXP3 mediates these effects. Using novel cell lines that inducibly express either wild-type or mutant FOXP3, we have identified NFAT2 as an early target of FOXP3-mediated transcriptional repression. NFAT2 is typically expressed at low levels in resting T cells, but is up-regulated by NFAT1 upon cellular activation. We demonstrate that transcription from the NFAT2 promoter is significantly suppressed by FOXP3, and NFAT2 protein expression is markedly diminished in activated CD4(+)CD25(+)FOXP3(+) T(REG) compared with CD4(+)CD25(-)FOXP3(-) T cells. Chromatin immunoprecipitation experiments indicate that FOXP3 competes with NFAT1 for binding to the endogenous NFAT2 promoter. This antagonism of NFAT2 activity by FOXP3 is important for the anergic phenotype of T(REG), as ectopic expression of NFAT2 from a retroviral LTR partially restores expression of IL-2 in FOXP3(+) T(REG). These data suggest that FOXP3 functions not only to suppress the first wave of NFAT-mediated transcriptional responses, but may also affect sustained NFAT-mediated inflammatory gene expression through suppression of inducible NFAT2 transcription.

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.

Analysis of genes differentially expressed during retinal degeneration in three mouse models.

An estimated 100,000 people in the US alone have retinitis pigmentosa. This disease, caused by the loss of rods and cones, results in blindness. With the intention of identifying common cell death pathways that result in RP, the pattern of global gene expression in three different mouse models of retinal degeneration was analyzed using DNA arrays. The models used were opsin ( Delta255-256 ), a transgenic mouse line that expresses a mutant form of opsin with a deletion of an isoleucine at either position 255 or 256; the Bouse C mouse, whereby normal opsin is over-expressed by over 2 folds; MOT1, a model that expresses SV-40 T antigen downstream of opsin promoter and leads to retinal degeneration. We found that, at least in the 2 models of retinal degeneration that are characterized by rhodopsin abnormalities, death is due to the TNF pathway. In addition, there are a number of unknown genes not yet annotated in each of the models that could be promising in revealing novel functions in photoreceptors.

Endothelial Activation Markers as Disease Activity and Damage Measures in Juvenile Dermatomyositis.

OBJECTIVE: Circulating endothelial cells (CEC), von Willebrand factor (vWF) antigen, P-selectin, and thrombomodulin are released from damaged endothelium, while decreases in circulating endothelial progenitor cells (CEPC) have been associated with poor vascular outcomes. We examined these markers in the peripheral blood of patients with juvenile dermatomyositis (JDM) and their correlations with disease assessments. METHODS: Peripheral blood endothelial cells and biomarkers were assessed in 20 patients with JDM and matched healthy controls. CEC and CEPC were measured by flow cytometry, while vWF antigen and activity, factor VIII, P-selectin, and thrombomodulin were measured in plate-based assays. Disease activity and damage, nailfold capillary density, and brachial artery flow dilation were assessed. Serum cytokines/chemokines were measured by Luminex. RESULTS: CEC, vWF antigen, factor VIII, and thrombomodulin, but not vWF activity, CEPC, or P-selectin, were elevated in the peripheral blood of patients with JDM. CEC correlated with pulmonary activity (rs = 0.56). The vWF antigen correlated with Patient's/Parent's Global, cutaneous, and extramuscular activity (rs = 0.47-0.54). CEPC negatively correlated with muscle activity and physical function (rs = -0.52 to -0.53). CEPC correlated inversely with endocrine damage. The vWF antigen and activity correlated with interleukin 10 and interferon-gamma inducible protein-10 (rs = 0.64-0.82). CONCLUSION: Markers of endothelial injury are increased in patients with JDM and correlate with extramuscular activity. CEPC correlate inversely with muscle activity, suggesting a functional disturbance in repair mechanisms.

Independent Validation for the Polyskope 1.0 Multiplex Pathogen Detection Assay for the Detection of Shiga Toxin-Producing Escherichia coli Non-O157 STEC, Escherichia coli O157, Listeria monocytogenes, and Salmonella Species.

BACKGROUND: The Polyskope 1.0 Multiplex Assay is a novel test to simultaneously detect Escherichia coli O157, non-O157 Shiga Toxin-Producing E. coli (STEC), Listeria monocytogenes, and Salmonella species in a single enrichment using real-time PCR. OBJECTIVE: A Performance Tested MethodSM study was conducted to validate Polyskope 1.0 for inclusivity and exclusivity as well as a matrix comparison study. METHOD: This assay was evaluated in an unpaired independent validation study compared with reference methods according to AOAC INTERNATIONAL validation guidelines. Polyskope 1.0 evaluated raw ground beef (25 g), deli turkey (25 g), baby spinach (25 g), and stainless-steel environmental surface sponges (4 × 4 in. test area) after inoculation with a suspension of the three target microorganisms. All matrices were compared with appropriate reference methods from the U.S. Food and Drug Administration Bacteriological Analytical Manual, U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook, or International Organization for Standardization standards. RESULTS: Polyskope 1.0 demonstrated no statistically significant differences between candidate and reference method results or between presumptive and confirmed results for three food matrices and one environmental surface. Results from inclusivity and exclusivity evaluations indicated the test method can accurately detect the target analytes and excluded all nontarget organisms. No differences were observed with the stability or lot-to-lot evaluations. Polyskope 1.0 demonstrated robustness by remaining unaffected by small variations in method parameters, which had no statistically significant effect on the results for all eight variations. Conclusions and Highlights: Polyskope 1.0 was shown to be a specific, highly accurate, and robust method for the detection of Listeria monocytogenes, Salmonella species, non-O157 STECs, and E. coli O157 across four matrices.

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.

Temporal dynamics of gene expression in the lung in a baboon model of E. coli sepsis.

BACKGROUND: Bacterial invasion during sepsis induces disregulated systemic responses that could lead to fatal lung failure. The purpose of this study was to relate the temporal dynamics of gene expression to the pathophysiological changes in the lung during the first and second stages of E. coli sepsis in baboons. RESULTS: Using human oligonucleotide microarrays, we have explored the temporal changes of gene expression in the lung of baboons challenged with sublethal doses of E. coli. Temporal expression pattern and biological significance of the differentially expressed genes were explored using clustering and pathway analysis software. Expression of selected genes was validated by real-time PCR. Cytokine levels in tissue and plasma were assayed by multiplex ELISA. Changes in lung ultrastructure were visualized by electron microscopy. We found that genes involved in primary inflammation, innate immune response, and apoptosis peaked at 2 hrs. Inflammatory and immune response genes that function in the stimulation of monocytes, natural killer and T-cells, and in the modulation of cell adhesion peaked at 8 hrs, while genes involved in wound healing and functional recovery were upregulated at 24 hrs. CONCLUSION: The analysis of gene expression modulation in response to sepsis provides the baseline information that is crucial for the understanding of the pathophysiology of systemic inflammation and may facilitate the development of future approaches for sepsis therapy.

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.

Influence of intraarticular corticosteroid administration on serum cytokines in rheumatoid arthritis.

Though the efficacy of intraarticular (IA) corticosteroid administration in the therapeutic management of rheumatoid arthritis (RA) has been well documented, its immunomodulatory effects have not been defined. Categorization of these effects is important to develop safe derivative therapeutic strategies with a more targeted mechanism of action as they relate to the pathophysiology of RA. We describe here a broad spectrum immune response to inflammation as evidenced by rapid transient systemic inhibitory effects on key inflammatory regulators induced by the effects of IA administration of triamcinolone acetonide in a case of active RA who failed to respond to methotrexate.