The dynamics of signaling as a pharmacological target.

Highly networked signaling hubs are often associated with disease, but targeting them pharmacologically has largely been unsuccessful in the clinic because of their functional pleiotropy. Motivated by the hypothesis that a dynamic signaling code confers functional specificity, we investigated whether dynamic features may be targeted pharmacologically to achieve therapeutic specificity. With a virtual screen, we identified combinations of signaling hub topologies and dynamic signal profiles that are amenable to selective inhibition. Mathematical analysis revealed principles that may guide stimulus-specific inhibition of signaling hubs, even in the absence of detailed mathematical models. Using the NFκB signaling module as a test bed, we identified perturbations that selectively affect the response to cytokines or pathogen components. Together, our results demonstrate that the dynamics of signaling may serve as a pharmacological target, and we reveal principles that delineate the opportunities and constraints of developing stimulus-specific therapeutic agents aimed at pleiotropic signaling hubs.

Evaluation of novel serological markers and autoantibodies in dogs with inflammatory bowel disease.

BACKGROUND: The use of serological markers to diagnose inflammatory bowel disease (IBD) in humans is well-established. Because of the frequency of IBD in dogs and resources required for its diagnosis with current methods, new approaches are desired. OBJECTIVE: The goal is to evaluate novel serologic markers to differentiate clinical cohorts in dogs with gastrointestinal (GI) disease and assess their potential to develop a serum-based IBD diagnostic test. ANIMALS: Seventy dogs diagnosed with biopsy-confirmed IBD, 23 dogs with non-IBD predominantly acute GI diseases, and 58 normal dogs. METHODS: Prospective control study. ELISA methods were developed to detect autoantibodies to polymorphonuclear leukocytes (APMNA) and calprotectin (ACNA), antibodies against gliadins (AGA), microbial outer membrane porin C (ACA), and flagellins (AFA) isolated from diseased dogs based on clinical and histopathological scoring. RESULTS: IBD dogs displayed a 39%-76% prevalence of seropositivity against selected serologic markers that markedly decreased to 0%-13% in non-IBD and normal dogs. ROC analysis showed statistical significance in differentiating the cohorts, with seropositivity against OmpC being the highest single performance marker. The combination of markers such as OmpC and APMNA reached specificities of 93%-99% and 79%-98% and sensitivities of 76%-97% and 66%-86% when comparing IBD versus normal cohorts and non-IBD cohorts, respectively. CONCLUSION AND CLINICAL IMPORTANCE: Seropositivity of canine immunoglobulins A against selected serologic markers in dogs appears promising in the detection and differentiation of IBD versus other acute GI conditions. Among them, antibody reactivity to Escherichia coli OmpC and canine autoantibodies against polymorphonuclear leukocytes displayed the highest single marker discriminating performance.

A homeostatic model of IkappaB metabolism to control constitutive NF-kappaB activity.

Cellular signal transduction pathways are usually studied following administration of an external stimulus. However, disease-associated aberrant activity of the pathway is often due to misregulation of the equilibrium state. The transcription factor NF-kappaB is typically described as being held inactive in the cytoplasm by binding its inhibitor, IkappaB, until an external stimulus triggers IkappaB degradation through an IkappaB kinase-dependent degradation pathway. Combining genetic, biochemical, and computational tools, we investigate steady-state regulation of the NF-kappaB signaling module and its impact on stimulus responsiveness. We present newly measured in vivo degradation rate constants for NF-kappaB-bound and -unbound IkappaB proteins that are critical for accurate computational predictions of steady-state IkappaB protein levels and basal NF-kappaB activity. Simulations reveal a homeostatic NF-kappaB signaling module in which differential degradation rates of free and bound pools of IkappaB represent a novel cross-regulation mechanism that imparts functional robustness to the signaling module.

Systemic lupus erythematosus and primary fibromyalgia can be distinguished by testing for cell-bound complement activation products.

OBJECTIVE: We sought to establish the performance of cell-bound complement activation products (CB-CAPs) as a diagnostic tool to distinguish primary fibromyalgia (FM) from systemic lupus erythematosus (SLE). METHODS: A total of 75 SLE and 75 primary FM adult subjects who fulfilled appropriate classification criteria were enrolled prospectively. CB-CAPs (erythrocyte-C4d (EC4d) and B-lymphocyte-C4d (BC4d)) were determined by flow cytometry. Antinuclear antibodies (ANAs) were determined using indirect immunofluorescence while other autoantibodies were determined by solid-phase assays. The CB-CAPs in a multi-analyte assay with algorithm (MAAA) relied on two consecutive tiers of analysis that was reported as an overall positive or negative assessment. Test performance was assessed using sensitivity, specificity, positive and negative likelihood ratio (LR). RESULTS: ANAs yielded 80% positives for SLE and 33% positives for FM. High CB-CAP expression (EC4d >14 units or BC4d >60 units) was 43% sensitive and 96% specific for SLE. The CB-CAPs in MAAA assessment was evaluable in 138 of the 150 subjects enrolled (92%) and yielded 60% sensitivity (CI 95% 48% to 72%) for SLE with no FM patient testing positive (100% specificity). A positive test result was associated with a strong positive LR for SLE (>24, CI 95%; 6 to 102), while a negative test result was associated with a moderate negative LR (0.40; CI 95% 0.30 to 0.54). CONCLUSION: Our data indicate that CB-CAPs in MAAA can distinguish FM from SLE.

The Avise Lupus Test and Cell-bound Complement Activation Products Aid the Diagnosis of Systemic Lupus Erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a multifaceted disease, and its diagnosis may be challenging. A blood test for the diagnosis of SLE, the Avise Lupus test, has been recently commercialized and validated in clinical studies. OBJECTIVES: To evaluate the use of the Avise Lupus test by community rheumatologists. METHODS: The study is a longitudinal, case-control, retrospective review of medical charts. Cases had a positive test result, and controls had a negative result; all patients were anti-nuclear antibodies (ANA) positive but negative for SLE-specific autoantibodies. Features of SLE, diagnosis, and medications at two time points were recorded. RESULTS: Twenty of the 23 cases (87%) and 4 of the 23 controls (17%) were diagnosed with SLE (sensitivity=83%; specificity=86%). More cases than controls (43% vs. 17%) fulfilled 4 American College of Rheumatology (ACR) classification criteria of SLE. Sensitivity of the test was significantly higher than the ACR score (83% vs. 42%, p=0.006). A higher percentage of patients who met the classification criteria had elevated cell-bound complement activation products (CB-CAPs) compared to patients who did not. Anti-rheumatic medications were used in a higher percentage of cases than controls (83% vs. 35% at baseline, p=0.002), suggesting that cases were treated more aggressively early on. CONCLUSION: A positive Avise Lupus test result aids in formulating a SLE diagnosis when diagnosis based on standard-of-care tests and clinical features may be challenging, and impacts patient management. Prospective studies will be performed to better evaluate the clinical utility of the test and of CB-CAPs as biomarkers of SLE.

Reduction in erythrocyte-bound complement activation products and titres of anti-C1q antibodies associate with clinical improvement in systemic lupus erythematosus.

BACKGROUND: The relationship between cell-bound complement activation products (CB-CAPs: EC4d, EC3d), anti-C1q, soluble complement C3/C4 and disease activity in systemic lupus erythematosus (SLE) was evaluated. METHODS: Per protocol, at baseline all SLE subjects enrolled in this longitudinal study presented with active disease and elevated CB-CAPs. At each monthly visit, the non-serological (ns) Safety of Estrogens in Lupus Erythematosus: National Assessment (SELENA-SLEDAI) and the British Isles Lupus Assessment Group (BILAG)-2004 index scores were determined as was a random urinary protein to creatinine ratio (uPCR). Short-form 36 (SF-36) questionnaires were also collected. All soluble markers were determined using immunoassays, while EC4d and EC3d were determined using flow cytometry. Statistical analysis consisted of linear mixed models with random intercept and fixed slopes. RESULTS: A total of 36 SLE subjects (mean age 34 years; 94% female) were enrolled and evaluated monthly for an average 11 visits per subject. Clinical improvements were observed during the study, with significant decreases in ns-SELENA-SLEDAI scores, BILAG-2004 index scores and uPCR, and increases in all domains of SF-36 (p<0.01). The longitudinal decrease in ns-SELENA-SLEDAI and BILAG-2004 index scores was significantly associated with reduced EC4d and EC3d levels, reduced anti-C1q titres and increased serum complement C3/C4 (p<0.05). The changes in uPCR significantly correlated with C3, C4, anti-C1q and EC4d, with EC4d outperforming C3/C4 by a multivariate analysis. The reduced EC4d or EC3d was associated with improvements in at least six out of the eight domains of SF-36 and outperformed C3/C4. Anti-dsDNA titres did not correlate with changes in disease activity. CONCLUSIONS: These data indicate that CB-CAPs and anti-C1q are helpful in monitoring patients with SLE.

Cell-bound complement activation products in systemic lupus erythematosus: comparison with anti-double-stranded DNA and standard complement measurements.

OBJECTIVE: To compare the performance characteristics of cell-bound complement (C4d) activation products (CBCAPS) on erythrocyte (EC4d) and B cells (BC4d) with antibodies to double-stranded DNA (anti-dsDNA) and complement C3 and C4 in systemic lupus erythematosus (SLE). METHODS: The study enrolled 794 subjects consisting of 304 SLE and a control group consisting of 285 patients with other rheumatic diseases and 205 normal individuals. Anti-dsDNA and other autoantibodies were measured using solid-phase immunoassays while EC4d and BC4d were determined using flow cytometry. Complement proteins were determined using immunoturbidimetry. Disease activity in SLE was determined using a non-serological Systemic Lupus Erythematosus Disease Activity Index SELENA Modification. A two-tiered methodology combining CBCAPS with autoantibodies to cellular and citrullinated antigens was also developed. Statistical analyses used area under receiver operating characteristic curves and calculations of area under the curve (AUC), sensitivity and specificity. RESULTS: AUC for EC4d (0.82±0.02) and BC4d (0.84±0.02) was higher than those yielded by C3 (0.73±0.02) and C4 (0.72±0.02) (p<0.01). AUC for CBCAPS was also higher than the AUC yielded by anti-dsDNA (0.79±0.02), but significance was only achieved for BC4d (p<0.01). The combination of EC4d and BC4d in multivariate testing methodology with anti-dsDNA and autoantibodies to cellular and citrullinated antigens yielded 80% sensitivity for SLE and specificity ranging from 70% (Sjogren's syndrome) to 92% (rheumatoid arthritis) (98% vs. normal). A higher proportion of patients with SLE with higher levels of disease activity tested positive for elevated CBCAPS, reduced complement and anti-dsDNA (p<0.03). CONCLUSIONS: CBCAPS have higher sensitivity than standard complement and anti-dsDNA measurements, and may help with the differential diagnosis of SLE in combination with other autoantibodies.

Combination of genetic and quantitative serological immune markers are associated with complicated Crohn's disease behavior.

BACKGROUND: Treatment of Crohn's disease (CD) with biologics may alter disease progression, leading to fewer disease-related complications, but cost and adverse event profiles often limit their effective use. Tools identifying patients at high risk of complications, who would benefit the most from biologics, would be valuable. Previous studies suggest that biomarkers may aid in determining the course of CD. We aimed to determine if combined serologic immune responses and NOD2 genetic markers are associated with CD complications. METHODS: In this cross-sectional study, banked blood from well-characterized CD patients (n = 593; mean follow-up: 12 years) from tertiary and community centers was analyzed for six serological biomarkers (ASCA-IgA, ASCA-IgG, anti-OmpC, anti-CBir1, anti-I2, pANCA). In a patient subset (n = 385), NOD2 (SNP8, SNP12, SNP13) genotyping was performed. Complications included stricturing and penetrating disease behaviors. A logistic regression model for the risk of complications over time was constructed and evaluated by cross-validation. RESULTS: For each serologic marker, complication rates were stratified by quartile. Complication frequency was significantly different across quartiles for each marker (P trend ≤ 0.001). Patients with SNP13 NOD2 risk alleles experienced increased complications versus patients without NOD2 mutations (P ≤ 0.001). A calibration plot of modeled versus observed complication rates demonstrated good agreement (R = 0.973). Performance of the model integrating serologic and genetic markers was demonstrated by area under the receiver operating characteristic curve (AUC = 0.801; 95% confidence interval: 0.757-0.846). CONCLUSIONS: This model combining serologic and NOD2 genetic markers may provide physicians with a tool to assess the probability of patients developing a complication over the course of CD.

Stimulus specificity of gene expression programs determined by temporal control of IKK activity.

A small number of mammalian signaling pathways mediate a myriad of distinct physiological responses to diverse cellular stimuli. Temporal control of the signaling module that contains IkappaB kinase (IKK), its substrate inhibitor of NF-kappaB (IkappaB), and the key inflammatory transcription factor NF-kappaB can allow for selective gene activation. We have demonstrated that different inflammatory stimuli induce distinct IKK profiles, and we examined the underlying molecular mechanisms. Although tumor necrosis factor-alpha (TNFalpha)-induced IKK activity was rapidly attenuated by negative feedback, lipopolysaccharide (LPS) signaling and LPS-specific gene expression programs were dependent on a cytokine-mediated positive feedback mechanism. Thus, the distinct biological responses to LPS and TNFalpha depend on signaling pathway-specific mechanisms that regulate the temporal profile of IKK activity.