BackgroundDisease activity measurement is a key component of rheumatoid arthritis (RA) management. Biomarkers that capture the complex and heterogeneous biology of RA have the potential to complement clinical disease activity assessment.ObjectivesTo develop a multi-biomarker disease activity (MBDA) test for rheumatoid arthritis.MethodsCandidate serum protein biomarkers were selected from extensive literature screens, bioinformatics databases, mRNA expression and protein microarray data. Quantitative assays were identified and optimized for measuring candidate biomarkers in RA patient sera. Biomarkers with qualifying assays were prioritized in a series of studies based on their correlations to RA clinical disease activity (e.g. the Disease Activity Score 28-C-Reactive Protein [DAS28-CRP], a validated metric commonly used in clinical trials) and their contributions to multivariate models. Prioritized biomarkers were used to train an algorithm to measure disease activity, assessed by correlation to DAS and area under the receiver operating characteristic curve for classification of low vs. moderate/high disease activity. The effect of comorbidities on the MBDA score was evaluated using linear models with adjustment for multiple hypothesis testing.Results130 candidate biomarkers were tested in feasibility studies and 25 were selected for algorithm training. Multi-biomarker statistical models outperformed individual biomarkers at estimating disease activity. Biomarker-based scores were significantly correlated with DAS28-CRP and could discriminate patients with low vs. moderate/high clinical disease activity. Such scores were also able to track changes in DAS28-CRP and were significantly associated with both joint inflammation measured by ultrasound and damage progression measured by radiography. The final MBDA algorithm uses 12 biomarkers to generate an MBDA score between 1 and 100. No significant effects on the MBDA score were found for common comorbidities.ConclusionWe followed a stepwise approach to develop a quantitative serum-based measure of RA disease activity, based on 12-biomarkers, which was consistently associated with clinical disease activity levels.

Although strong epidemiologic evidence suggests an important role for adaptive immunity in the pathogenesis of polyarticular juvenile rheumatoid arthritis (JRA), there remain many aspects of the disease that suggest equally important contributions of the innate immune system. We used gene expression arrays and computer modeling to examine the function in neutrophils of 25 children with polyarticular JRA. Computer analysis identified 712 genes that were differentially expressed between patients and healthy controls. Computer-assisted analysis of the differentially expressed genes demonstrated functional connections linked to both interleukin (IL)-8-and interferon-γ (IFN-γ)-regulated processes. Of special note is that the gene expression fingerprint of children with active JRA remained essentially unchanged even after they had responded to therapy. This result differed markedly from our previously reported work, in which gene expression profiles in buffy coats of children with polyarticular JRA reverted to normal after disease control was achieved pharmacologically. These findings suggest that JRA neutrophils remain in an activated state even during disease quiescence. Computer modeling of array data further demonstrated disruption of gene regulatory networks in clusters of genes modulated by IFN-γ and IL-8. These cytokines have previously been shown to independently regulate the frequency (IFN-γ) and amplitude (IL-8) of the oscillations of key metabolites in neutrophils, including nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and superoxide ion. Using real-time, high-speed, single-cell photoimaging, we observed that 6/6 JRA patients displayed a characteristic defect in 12% to 23% of the neutrophils tested. Reagents known to induce only frequency fluctuations of NAD(P)H and superoxide ion induced both frequency and amplitude fluctuations in JRA neutrophils. This is a novel finding that was observed in children with both active (n = 4) and inactive (n = 2) JRA. A subpopulation of polyarticular JRA neutrophils are in a chronic, activated state, a state that persists when the disease is well controlled pharmacologically. Furthermore, polyarticular JRA neutrophils exhibit an intrinsic defect in the regulation of metabolic oscillations and superoxide ion production. Our data are consistent with the hypothesis that neutrophils play an essential role in the pathogenesis of polyarticular JRA.

These results outline aspects of the multifaceted role B-cells play in RA pathogenesis in which immune dysregulation in RA modulates B-cell biology and thereby contributes to the induction and perpetuation of a pathogenic humoral immune response.

BackgroundAn 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.ResultsIt 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-κB 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.ConclusionA 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.