Aptamer Proteomics for Biomarker Discovery in Heart Failure With Preserved Ejection Fraction: The PARAGON-HF Proteomic Substudy.

BACKGROUND: Prognostic markers and biological pathways linked to detrimental clinical outcomes in heart failure with preserved ejection fraction (HFpEF) remain incompletely defined. METHODS AND RESULTS: We measured serum levels of 4123 unique proteins in 1117 patients with HFpEF enrolled in the PARAGON-HF (Efficacy and Safety of LCZ696 Compared to Valsartan, on Morbidity and Mortality in Heart Failure Patients With Preserved Ejection Fraction) trial using a modified aptamer proteomic assay. Baseline circulating protein concentrations significantly associated with the primary end point and the timing and occurrence of total heart failure hospitalization and cardiovascular death were identified by recurrent events regression, accounting for multiple testing, adjusted for age, sex, treatment, and anticoagulant use, and compared with published analyses in 2515 patients with heart failure with reduced ejection fraction from the PARADIGM-HF (Prospective Comparison of ARNI With ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure) and ATMOSPHERE (Efficacy and Safety of Aliskiren and Aliskiren/Enalapril Combination on Morbidity-Mortality in Patients With Chronic Heart Failure) clinical trials. We identified 288 proteins that were robustly associated with the risk of heart failure hospitalization and cardiovascular death in patients with HFpEF. The baseline proteins most strongly related to outcomes included B2M (ß-2 microglobulin), TIMP1 (tissue inhibitor of matrix metalloproteinase 1), SERPINA4 (serpin family A member 4), and SVEP1 (sushi, von Willebrand factor type A, EGF, and pentraxin domain containing 1). Overall, the protein-outcome associations in patients with HFpEF did not markedly differ as compared with patients with heart failure with reduced ejection fraction. A proteomic risk score derived in patients with HFpEF was not superior to a previous proteomic score derived in heart failure with reduced ejection fraction nor to clinical risk factors, NT-proBNP (N-terminal pro-B-type natriuretic peptide), or high-sensitivity cardiac troponin. CONCLUSIONS: Numerous serum proteins linked to metabolic, coagulation, and extracellular matrix regulatory pathways were associated with worse HFpEF prognosis in the PARAGON-HF proteomic substudy. Our results demonstrate substantial similarities among serum proteomic risk markers for heart failure hospitalization and cardiovascular death when comparing clinical trial participants with heart failure across the ejection fraction spectrum. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique Identifiers: NCT01920711, NCT01035255, NCT00853658.

Effect of Clonal Hematopoiesis Mutations and Canakinumab Treatment on Incidence of Solid Tumors in the CANTOS Randomized Clinical Trial.

Clonal hematopoiesis (CH) is more common in older persons and has been associated with an increased risk of hematological cancers and cardiovascular diseases. The most common CH mutations occur in the DNMT3A and TET2 genes and result in increased pro-inflammatory signaling. The Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS, NCT01327846) evaluated the neutralizing anti-IL-1ß antibody canakinumab in 10,061 randomized patients with a history of myocardial infarction and persistent inflammation; DNA samples were available from 3,923 patients for targeted genomic sequencing. We examined the incidence of non-hematological malignancy by treatment assignment and CH mutations and estimated the cumulative incidence of malignancy events during trial follow-up. Patients with TET2 mutations treated with canakinumab had the lowest incidence of non-hematological malignancy across cancer types. The cumulative incidence of at least one reported malignancy was lower for patients with TET2 mutations treated with canakinumab vs those treated with placebo. These findings support a potential role for canakinumab in cancer prevention and provide evidence of IL-1ß blockade cooperating with CH mutations to modify the disease course.

Effects of IL-1ß inhibition on anemia and clonal hematopoiesis in the randomized CANTOS trial.

Canakinumab, a monoclonal antibody targeting proinflammatory cytokine interleukin-1ß (IL-1ß), improved hemoglobin levels while preventing recurrent cardiovascular events in the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS). This cardiovascular (CV) preventive effect was greater in patients with TET2 mutations associated with clonal hematopoiesis (CH). The current proteogenomic analysis aimed to understand the clinical response to canakinumab and underlying proteomic profiles in the context of CH and anemia. The analysis included 4595 patients from the CANTOS study who received either canakinumab or placebo and evaluated multiplexed proteomics (4785 proteins) using SomaScan and targeted deep sequencing for CH mutations. Incident anemia was more common in the presence of CH mutations but reduced by canakinumab treatment. Canakinumab treatment was significantly associated with higher hemoglobin increment in patients with concurrent CH mutations and anemia than patients with CH mutations without anemia or without CH mutations. Compared with those without CH mutations, the presence of CH mutations was associated with proteomic signatures of inflammation and defense response to infection, as well as markers of high-risk CV disease which was further enhanced by the presence of anemia. Canakinumab suppressed hepcidin, proinflammatory cytokines, myeloid activation, and complement pathways, and reversed pathologically deregulated pathways to a greater extent in patients with CH mutations and anemia. These molecular findings provide evidence of the clinical use of IL-1ß blockade and support further study of canakinumab for patients with concurrent anemia and CH mutations. This study was registered at www.clinicaltrials.gov as #NCT01327846.

Response to anti-IL17 therapy in inflammatory disease is not strongly impacted by genetic background.

Response to the anti-IL17 monoclonal antibody secukinumab is heterogeneous, and not all participants respond to treatment. Understanding whether this heterogeneity is driven by genetic variation is a key aim of pharmacogenetics and could influence precision medicine approaches in inflammatory diseases. Using changes in disease activity scores across 5,218 genotyped individuals from 19 clinical trials across four indications (psoriatic arthritis, psoriasis, ankylosing spondylitis, and rheumatoid arthritis), we tested whether genetics predicted response to secukinumab. We did not find any evidence of association between treatment response and common variants, imputed HLA alleles, polygenic risk scores of disease susceptibility, or cross-disease components of shared genetic risk. This suggests that anti-IL17 therapy is equally effective regardless of an individual's genetic background, a finding that has important implications for future genetic studies of biological therapy response in inflammatory diseases.

TET2-Driven Clonal Hematopoiesis and Response to Canakinumab: An Exploratory Analysis of the CANTOS Randomized Clinical Trial.

Importance: Clonal hematopoiesis of indeterminate potential (CHIP) is associated with increased risk of atherosclerotic cardiovascular disease, and mouse experiments suggest that CHIP related to Tet2 loss of function in myeloid cells accelerates atherosclerosis via augmented interleukin (IL) 1ß signaling. Objective: To assess whether individuals with CHIP have greater cardiovascular event reduction in response to IL-1ß neutralization in the Canankinumab Anti-inflammatory Thrombosis Outcomes Trial (CANTOS). Design, Setting, and Participants: This randomized clinical trial took place from April 2011 to June 2017 at more than 1000 clinical sites in 39 countries. Targeted deep sequencing of genes previously associated with CHIP in a subset of trial participants using genomic DNA prepared from baseline peripheral blood samples were analyzed. All participants had prior myocardial infarction and elevated high-sensitivity C-reactive protein level above 0.20 mg/dL. Analysis took place between June 2017 and December 2021. Interventions: Canakinumab, an anti-IL-1ß antibody, given at doses of 50, 150, and 300 mg once every 3 months. Main Outcomes and Measures: Major adverse cardiovascular events (MACE). Results: A total of 338 patients (8.6%) were identified in this subset with evidence for clonal hematopoiesis. As expected, the incidence of CHIP increased with age; the mean (SD) age of patients with CHIP was 66.3 (9.2) years and 61.5 (9.6) years in patients without CHIP. Unlike other populations that were not preselected for elevated C-reactive protein, in the CANTOS population variants in TET2 were more common than DNMT3A (119 variants in 103 patients vs 86 variants in 85 patients). Placebo-treated patients with CHIP showed a nonsignificant increase in the rate of MACE compared with patients without CHIP using a Cox proportional hazard model (hazard ratio, 1.32 [95% CI, 0.86-2.04]; P = .21). Exploratory analyses of placebo-treated patients with a somatic variant in either TET2 or DNMT3A (n = 58) showed an equivocal risk for MACE (hazard ratio, 1.65 [95% CI, 0.97-2.80]; P = .06). Patients with CHIP due to somatic variants in TET2 also had reduced risk for MACE while taking canakinumab (hazard ratio, 0.38 [95% CI, 0.15-0.96]) with equivocal difference compared with others (P for interaction = .14). Conclusions and Relevance: These results are consistent with observations of increased risk for cardiovascular events in patients with CHIP and raise the possibility that those with TET2 variants may respond better to canakinumab than those without CHIP. Future studies are required to further substantiate this hypothesis. Trial Registration: ClinicalTrials.gov Identifier: NCT01327846.

Endometrial cytokines in patients with and without endometriosis evaluated for infertility.

OBJECTIVE: To evaluate whether endometrial molecular profiles distinguish subsets of patients according to clinical characteristics, and to infer dysregulated immune networks, by measuring cytokines, chemokines, and growth factors in endometrial biopsy specimens from a cohort of infertile women with a high incidence of endometriosis. DESIGN: Prospective cohort study. SETTING: Department of Gynecology at a university hospital. PATIENT(S): Patients undergoing laparoscopy for infertility assessment (n = 103). INTERVENTION(S): Endometrial biopsies were performed during surgery. Fertility outcome and clinical parameters were registered preoperatively and after 6 months. MAIN OUTCOME MEASURE(S): The concentrations of 48 factors in endometrial biopsy specimens were analyzed with respect to clinical status in univariate and multivariate frameworks. RESULT(S): The concentrations of 44 factors from endometrial tissues of 74 patients were suitable for analysis. Although the tissue concentrations of interleukin (IL)15, IL-7, and interferon γ-induced protein (IP)-10 were individually lower in patients with endometriosis than in those without endometriosis, the differences were not significant after multiple comparison. However, multivariate modeling incorporating covariation showed separation between subsets of endometriotic and nonendometriotic patients, based predominantly on IP-10, monocyte chemoattractant protein-1, IL-16, and IL-18; this result was independent of cycle and fertility status. Analysis restricted to endometrial tissues from the secretory phase separated endometriotic and nonendometriotic patients by a combination of IL-15, IP-10, monocyte chemoattractant protein-1, IL-16, and IL-18. This combination suggests a uterine natural killer cell defect. We found no significant correlations between endometrial cytokines and fertility outcome. CONCLUSION(S): A molecular signature in endometrial tissue was able to distinguish endometriotic from nonendometriotic patients, implicating uterine natural killer cells in endometriosis.

Comparison of cytokines in the peritoneal fluid and conditioned medium of adolescents and adults with and without endometriosis.

PROBLEM: To compare inflammatory- and immune-associated peritoneal cytokines of adolescents and adults with and without endometriosis. METHODS OF STUDY: In a nested case-control study in multiple university-affiliated scientific centers, ten adolescents and thirteen adults with visually and histologically confirmed endometriosis (cases), thirteen adolescents with visually suspected endometriosis but indeterminate (seven patients) or negative (six patients) histology, and fifteen adults undergoing surgery for non-malignant gynecologic disease without endometriosis (controls) underwent laparoscopic aspiration of peritoneal fluid (PF), from which PF and conditioned medium (CM) cytokine levels were assayed. RESULTS: Compared to adults with endometriosis, MCP-3, IL-12p40, MIP-1ß, and IL-15 were significantly higher among adolescents with endometriosis, while TNF-ß and CTACK were lower among adolescents. These differences were similar comparing adolescents with endometriosis to adult controls except for MIP-1ß, which was not statistically different. MIP-1ß was, however, the only cytokine observed to differ between adult cases and controls. There were no significant differences in CM cytokines among the three groups. Results were similar when analyses were restricted to samples collected (a) during menstrual cycle days 1-10, (b) from patients unexposed to exogenous hormones, or (c) from all adolescents despite presence or absence of histologic endometriosis. CONCLUSION: Biologically relevant and statistically significant differences in six PF cytokines were observed and suggest a more pro-invasion cytokine profile among adolescents with endometriosis. Adolescents with endometriosis have unique peritoneal cytokine profiles and molecular behavior when compared to adults with and without endometriosis.

Inhibition of IL1ß by Canakinumab May Be Effective against Diverse Molecular Subtypes of Lung Cancer: An Exploratory Analysis of the CANTOS Trial.

In the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS), inhibition of the IL1ß inflammatory pathway by canakinumab has been shown to significantly reduce lung cancer incidence and mortality. Here we performed molecular characterization of CANTOS patients who developed lung cancer during the study, including circulating tumor DNA (ctDNA) and soluble inflammatory biomarker analysis. Catalogue of Somatic Mutations in Cancer (COSMIC) database ctDNA mutations were detected in 65% (46/71) of the CANTOS patients with lung cancer, with 51% (36/71) having detectable ctDNA at the time point closest to lung cancer diagnosis and 43% (29/67) having detectable ctDNA at trial randomization. Mutations commonly found in lung cancer were observed with no evidence of enrichment in any mutation following canakinumab treatment. Median time to lung cancer diagnosis in patients with (n = 29) versus without (n = 38) detectable COSMIC ctDNA mutations at baseline was 407 days versus 837 days (P = 0.011). For serum inflammatory biomarker analysis, circulating levels of C-reactive protein (CRP), IL6, IL18, IL1 receptor antagonist, TNFα, leptin, adiponectin, fibrinogen, and plasminogen activator inhibitor-1 were determined. Patients with the highest level of baseline CRP or IL6, both downstream of IL1ß signaling, trended toward a shorter time to lung cancer diagnosis. Other inflammation markers outside of the IL1ß pathway at baseline did not trend with time to lung cancer diagnosis. These results provide further evidence for the importance of IL1ß-mediated protumor inflammation in lung cancer and suggest canakinumab's effect may be mediated in part by delaying disease progression of diverse molecular subtypes of lung cancer. SIGNIFICANCE: These findings suggest that targeting the IL1ß inflammatory pathway might be critical in reducing tumor-promoting inflammation and lung cancer incidence.

Endometrioma, the follicular fluid inflammatory network and its association with oocyte and embryo characteristics.

RESEARCH QUESTION: What is the association between endometrioma-affected ovaries, their follicular fluid inflammatory microenvironment, and ovary-specific oocyte and embryo yield and quality? DESIGN: Exposure-matched prospective cohort study conducted at a university-affiliated infertility clinic. Thirty-four women presenting for oocyte retrieval were enrolled between 2012 and 2013: women with unilateral endometrioma and no other observed peritoneal or deep lesions (n = 10) and women with no signs or symptoms of endometriosis (n = 24). Follicular fluid was aspirated at the time of oocyte retrieval. Samples from each ovary were analysed using a 27-plex immunoassay panel. The associations were evaluated by ovary-specific endometrioma exposure status (affected, unaffected, unexposed) with cytokine levels, oocyte yield and embryo quality. RESULTS: Levels of interleukin (IL)-8 and monocyte chemoattractant protein-1 were higher in fluid obtained from endometrioma-affected ovaries compared with the unexposed ovaries from women without endometriosis, with intermediate levels observed in the contralateral unaffected ovaries. More modest differences were observed for IL-1ß and IL-6. The affected ovaries of women with endometriosis yielded fewer oocytes (mean ± SD = 4.6 ± 2.3) compared with both the unaffected (6.0 ± 3.8) and unexposed (7.9 ± 5.6) ovaries. After adjusting for potential confounders and variables generated in a cytokine principal components analysis, oocyte yield remained slightly lower for the endometrioma-affected ovaries compared with unexposed ovaries. No informative differences among ovary groups for embryo quality parameters were observed. CONCLUSIONS: The results suggest that the inflammatory milieu of ovarian endometriosis is strongly localized and has a more modestly systemic effect. The effect of endometriomas on infertility, however, cannot be entirely explained by increased inflammation.

Identifying Key Pathways and Components in Chemokine-Triggered T Lymphocyte Arrest Dynamics Using a Multi-Parametric Global Sensitivity Analysis.

INTRODUCTION: The arrest of rolling T lymphocytes at specific locations is crucial to proper immune response function. We previously developed a model of chemokine-driven integrin activation, termed integrative signaling adhesive dynamics (ISAD). In addition, we have shown that loss of diacylglycerol kinase (DGK) leads to a gain of function regarding adhesion under shear flow. We undertook this study to understand the sensitivity of adhesion to perturbations in other signaling molecules. METHODS: We adapted multi-parametric sensitivity analysis (MPSA) for use in our ISAD model to identify important parameters, including initial protein concentrations and kinetic rate constants, for T lymphocyte arrest. We also compared MPSA results to those obtained from a single parametric sensitivity analysis. RESULTS: In addition to the previously shown importance of DGK in lymphocyte arrest, PIP2 cleavage and Rap1 activation are crucial in determining T cell arrest dynamics, which agree with previous experimental findings. The l-selectin density on the T lymphocyte surface also plays a large role in determining the distance rolled before arrest. Both the MPSA and single-parametric method returned similar results regarding the most sensitive kinetic rate constants. CONCLUSION: We show here that the regulation of the amount of second messengers are, in general, more critical for determining T lymphocyte arrest over the initial signaling proteins, highlighting the importance of amplification of signaling in cell adhesion responses. Overall, this work provides a mechanistic insight of the contribution of key pathways and components, thus may help to identify potential therapeutic targets for drug development against immune disorders.

Omalizumab normalizes the gene expression signature of lesional skin in patients with chronic spontaneous urticaria: A randomized, double-blind, placebo-controlled study.

BACKGROUND: Omalizumab, a humanized recombinant monoclonal anti-IgE antibody, proved to be effective in patients with chronic spontaneous urticaria (CSU), including severe and treatment-refractory CSU. Here, we report omalizumab's effect on gene expression in skin biopsies from CSU patients enrolled in a double-blind, placebo-controlled study. METHODS: Chronic spontaneous urticaria patients (18-75 years) were randomized to either 300 mg omalizumab (n = 20) or placebo (n = 10) administered s.c. every 4 weeks for 12 weeks (NCT01599637). Lesional and nonlesional skin biopsies were collected from the same area of consenting patients and assessed at baseline and on Day 85 compared with skin biopsies from the same area of 10 untreated healthy volunteers (HVs). Gene expression data were generated using Affymetrix HG-U133Plus2.0 microarrays. Statistical analyses were performed using R packages. RESULTS: At baseline, 63 transcripts were differentially expressed between lesional and nonlesional skin. Two-thirds of these lesional signatures were also differentially expressed between lesional and HV skin. Upon treatment with omalizumab, >75% of lesional signatures changed to reflect nonlesional skin expression levels (different vs placebo, P < 0.01). Transcripts upregulated in lesional skin (vs nonlesional and/or HV skin) suggested increased mast cell/leukocyte infiltration (FCER1G, C3AR1, CD93, S100A8, and S100A9), increased oxidative stress, vascularization (CYR61), and skin repair events (KRT6A, KRT16). Lesional signatures were not modulated by treatment in nonresponders (defined based on UAS7 longitudinal changes ≥16). CONCLUSION: Omalizumab, in treatment responders, reverted transcriptional signatures associated with CSU lesion phenotype to reflect nonlesional/HV expression levels; this is consistent with observed omalizumab-mediated clinical improvement observed in patients with CSU.

Large-scale identification of clinical and genetic predictors of motor progression in patients with newly diagnosed Parkinson's disease: a longitudinal cohort study and validation.

BACKGROUND: Better understanding and prediction of progression of Parkinson's disease could improve disease management and clinical trial design. We aimed to use longitudinal clinical, molecular, and genetic data to develop predictive models, compare potential biomarkers, and identify novel predictors for motor progression in Parkinson's disease. We also sought to assess the use of these models in the design of treatment trials in Parkinson's disease. METHODS: A Bayesian multivariate predictive inference platform was applied to data from the Parkinson's Progression Markers Initiative (PPMI) study (NCT01141023). We used genetic data and baseline molecular and clinical variables from patients with Parkinson's disease and healthy controls to construct an ensemble of models to predict the annual rate of change in combined scores from the Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) parts II and III. We tested our overall explanatory power, as assessed by the coefficient of determination (R2), and replicated novel findings in an independent clinical cohort from the Longitudinal and Biomarker Study in Parkinson's disease (LABS-PD; NCT00605163). The potential utility of these models for clinical trial design was quantified by comparing simulated randomised placebo-controlled trials within the out-of-sample LABS-PD cohort. FINDINGS: 117 healthy controls and 312 patients with Parkinson's disease from the PPMI study were available for analysis, and 317 patients with Parkinson's disease from LABS-PD were available for validation. Our model ensemble showed strong performance within the PPMI cohort (five-fold cross-validated R2 41%, 95% CI 35-47) and significant-albeit reduced-performance in the LABS-PD cohort (R2 9%, 95% CI 4-16). Individual predictive features identified from PPMI data were confirmed in the LABS-PD cohort. These included significant replication of higher baseline MDS-UPDRS motor score, male sex, and increased age, as well as a novel Parkinson's disease-specific epistatic interaction, all indicative of faster motor progression. Genetic variation was the most useful predictive marker of motor progression (2·9%, 95% CI 1·5-4·3). CSF biomarkers at baseline showed a more modest (0·3%, 95% CI 0·1-0·5) but still significant effect on prediction of motor progression. The simulations (n=5000) showed that incorporating the predicted rates of motor progression (as assessed by the annual change in MDS-UPDRS score) into the final models of treatment effect reduced the variability in the study outcome, allowing significant differences to be detected at sample sizes up to 20% smaller than in naive trials. INTERPRETATION: Our model ensemble confirmed established and identified novel predictors of Parkinson's disease motor progression. Improvement of existing prognostic models through machine-learning approaches should benefit trial design and evaluation, as well as clinical disease monitoring and treatment. FUNDING: Michael J Fox Foundation for Parkinson's Research and National Institute of Neurological Disorders and Stroke.

Peritoneal fluid cytokines related to endometriosis in patients evaluated for infertility.

OBJECTIVE: Our aim was to characterize peritoneal cytokine profiles in patients with infertility, with and without endometriosis, to illuminate potential differences in immune profiles that may reflect mechanistic differences between these two patient populations. DESIGN: Cross-sectional study. SETTING: University hospital and research center. PATIENT(S): Women undergoing laparoscopy for infertility investigation (n = 107). INTERVENTION(S): Peritoneal fluid was collected during surgery. Clinical characteristics were registered preoperatively. MAIN OUTCOME MEASURE(S): We determined the concentration of 48 different cytokines from the peritoneal fluid with multiplex immunoassays. Associations between cytokines and clinical findings were assessed with logistic regression and partial least squares discriminant analyses (PLS-DA). RESULT(S): Concentrations of SCGF-ß, IL-8, HGF, and MCP-1 were significantly higher, while IL-13 was significantly lower in the endometriosis group compared with the group without endometriosis. Multiple stepwise logistic regression identified a combination of SCGF-ß, IL-13, and G-CSF concentrations that predicted the presence of endometriosis with 86% sensitivity and 67% specificity. PLS-DA identified a class of 11 cytokines (SCGF-ß, HGF, IL-13, MCP-1, CTACK, MCP-3, M-CSF, LIF, IL-5, IL-9, and IFN-a2) that were more characteristic of endometriosis than nonendometriosis patients. CONCLUSION(S): By combining univariate and multivariate analyses, profiles of cytokines more likely to be enriched or depleted in infertility patients with endometriosis compared with those without endometriosis were identified. These findings may inform future analyses of pathophysiological mechanisms of endometriosis in infertile patients, including dysregulated Th1/Th2 response and mobilization and proliferation of hematopoietic stem cells.

Immobilized IL-8 Triggers Phagocytosis and Dynamic Changes in Membrane Microtopology in Human Neutrophils.

The interaction of leukocytes with surface bound ligands can be limited by the location of the molecules relative to the surface topology of the cell. In this report, we examine the dynamic response of neutrophils to IL-8-fractalkine chimera immobilized on bead surfaces, taking into account changes in receptor occupancy resulting from changes in surface topography. As a readout for receptor signaling, we observe the dynamics of calcium release in neutrophils following contact with the IL-8 coated surface. After a delay that depended on the initial area of contact and the surface density of IL-8, the cell began to phagocytose the IL-8 coated bead. This appeared to be a pre-requisite for release of calcium, which typically followed shortly after the initiation of phagocytosis. In separate experiments, effective kinetic coefficients for the formation of bonds between immobilized IL-8 and receptors on the cell surface were determined. Using these coefficients, we were able to estimate the number of bound receptors in the nascent contact zone. Kinetic modeling of the signaling response predicted that cell spreading and a concomitant increase in the density of occupied receptors would be required for the experimentally observed calcium dynamics. Postulating that there is an increase in receptor occupancy resulting from smoothing of the cell surface as it is stretched over the bead enabled us to obtain model predictions consistent with experimental observations. This study reveals the likely importance of membrane microtopology as a rate-limiting property and potential means of regulation of cell responses stimulated by two-dimensional surface interactions.

Molecular network analysis of endometriosis reveals a role for c-Jun-regulated macrophage activation.

Clinical management of endometriosis is limited by the complex relationship between symptom severity, heterogeneous surgical presentation, and variability in clinical outcomes. As a complement to visual classification schemes, molecular profiles of disease activity may improve risk stratification to better inform treatment decisions and identify new approaches to targeted treatment. We use a network analysis of information flow within and between inflammatory cells to discern consensus behaviors characterizing patient subpopulations. Unsupervised multivariate analysis of cytokine profiles quantified by multiplex immunoassays identified a subset of patients with a shared "consensus signature" of 13 elevated cytokines that was associated with common clinical features of endometriosis, but was not observed among patient subpopulations defined by morphologic presentation alone. Enrichment analysis of consensus markers reinforced the primacy of peritoneal macrophage infiltration and activation, which was demonstrably elevated in ex vivo cultures. Although familiar targets of the nuclear factor κB family emerged among overrepresented transcriptional binding sites for consensus markers, our analysis provides evidence for an unexpected contribution from c-Jun, c-Fos, and AP-1 effectors of mitogen-associated kinase signaling. Their crucial involvement in propagation of macrophage-driven inflammatory networks was confirmed via targeted inhibition of upstream kinases. Collectively, these analyses suggest a clinically relevant inflammatory network that may serve as an objective measure for guiding treatment decisions for endometriosis management, and in the future may provide a mechanistic endpoint for assessing efficacy of new agents aimed at curtailing inflammatory mechanisms that drive disease progression.

ADAM-10 and -17 regulate endometriotic cell migration via concerted ligand and receptor shedding feedback on kinase signaling.

A Disintegrin and Metalloproteinases (ADAMs) are the principal enzymes for shedding receptor tyrosine kinase (RTK) ectodomains and ligands from the cell surface. Multiple layers of activity regulation, feedback, and catalytic promiscuity impede our understanding of context-dependent ADAM "sheddase" function and our ability to predictably target that function in disease. This study uses combined measurement and computational modeling to examine how various growth factor environments influence sheddase activity and cell migration in the invasive disease of endometriosis. We find that ADAM-10 and -17 dynamically integrate numerous signaling pathways to direct cell motility. Data-driven modeling reveals that induced cell migration is a quantitative function of positive feedback through EGF ligand release and negative feedback through RTK shedding. Although sheddase inhibition prevents autocrine ligand shedding and resultant EGF receptor transactivation, it also leads to an accumulation of phosphorylated receptors (HER2, HER4, and MET) on the cell surface, which subsequently enhances Jnk/p38 signaling. Jnk/p38 inhibition reduces cell migration by blocking sheddase activity while additionally preventing the compensatory signaling from accumulated RTKs. In contrast, Mek inhibition reduces ADAM-10 and -17 activities but fails to inhibit compensatory signaling from accumulated RTKs, which actually enhances cell motility in some contexts. Thus, here we present a sheddase-based mechanism of rapidly acquired resistance to Mek inhibition through reduced RTK shedding that can be overcome with rationally directed combination inhibitor treatment. We investigate the clinical relevance of these findings using targeted proteomics of peritoneal fluid from endometriosis patients and find growth-factor-driven ADAM-10 activity and MET shedding are jointly dysregulated with disease.

Multiplexed protease activity assay for low-volume clinical samples using droplet-based microfluidics and its application to endometriosis.

As principal degrading enzymes of the extracellular matrix, metalloproteinases (MPs) contribute to various pathologies and represent a family of promising drug targets and biomarker candidates. However, multiple proteases and endogenous inhibitors interact to govern MP activity, often leading to highly context-dependent protease function that unfortunately has impeded associated clinical utility. We present a method for rapidly assessing the activity of multiple specific proteases in small volumes (<20 µL) of complex biological fluids such as clinical samples that are available only in very limited amounts. It uses a droplet-based microfluidic platform that injects the sample into thousands of picoliter-scale droplets from a barcoded droplet library (DL) containing mixtures of unique, moderately selective FRET-based protease substrates and specific inhibitors and monitors hundreds of the reactions thus initiated simultaneously by tracking these droplets. Specific protease activities in the sample are then inferred from the reaction rates using a deconvolution technique, proteolytic activity matrix analysis (PrAMA). Using a nine-member DL with three inhibitors and four FRET substrates, we applied the method to the peritoneal fluid of subjects with and without the invasive disease endometriosis. The results showed clear and physiologically relevant differences with disease, in particular, decreased MMP-2 and ADAM-9 activities.

Diacylglycerol kinase zeta negatively regulates CXCR4-stimulated T lymphocyte firm arrest to ICAM-1 under shear flow.

T lymphocyte arrest within microvasculature is an essential process in immune surveillance and the adaptive immune response. Integrins and chemokines coordinately regulate when and where T cells stop under flow via chemokine-triggered inside-out activation of integrins. Diacylglycerol kinases (DGKs) regulate the levels of diacylglycerol (DAG) which in turn determine the activation of guanine nucleotide exchange factors (GEFs) and Ras proximity 1 (Rap1) molecules crucial to the activation of integrin lymphocyte function-associated antigen 1 (LFA-1). However, how the level of DGK regulates chemokine-stimulated LFA-1-mediated T cell arrest under flow is unknown. Using a combination of experiment and computational modeling, we demonstrate that DGKζ is a crucial regulator of CXCL12-triggered T cell arrest on surfaces presenting inter-cellular adhesion molecule 1 (ICAM-1). Using flow chamber assays, we found that the deficiency of DGKζ in T cells significantly increased firm arrest to ICAM-1-coated substrates and shortened the time to stop without altering the rolling velocity. These results suggest that DGKζ levels affect LFA-1-mediated T cell firm arrest, but not P-selectin-mediated rolling during CXCL12 stimulation. We accurately simulated the role of DGKζ in firm arrest of T cells computationally using an Integrated-Signaling Adhesive Dynamics (ISAD). In the absence of DGK catalytic reaction, the model cells rolled for a significantly shorter time before arrest, compared to when DGK molecules were present. Predictions of our model for T cell arrest quantitatively match experimental results. Overall these results demonstrate that DGKζ is a negative regulator of CXCL12-triggered inside-out activation of LFA-1 and firm adhesion of T cells under shear flow.