Yael Rosenberg-Hasson

Effects of serum and plasma matrices on multiplex immunoassays.

Multiplexed fluorescence or electrochemiluminescence immunoassays of soluble cytokines are commonly performed in the context of human serum or plasma, to look for disease biomarkers and to monitor the immune system in a simple and minimally invasive way. These assays provide challenges due to the complexities of the matrix (serum or plasma) and the presence of many cytokines near the limit of detection of the assay. Here, we compare the readout of matched serum and plasma samples, which are generally correlated. However, a subset of cytokines usually have higher levels in serum, and the non-specific background is significantly increased in serum versus plasma. Presumably as a result of this non-specific background, disease-related decreases in low-abundance cytokines can sometimes be detected in plasma but not in serum. We further show, through spike recovery experiments, that both serum and plasma inhibit the readout of many cytokines, with some variability between donors, but with serum causing greater inhibition than plasma in many cases. Standard diluents from different vendors can partially reverse this inhibition to varying degrees. Dilution of samples can also partly overcome the inhibitory effect of the matrix. We also show that dilution is nonlinear and differentially affects various cytokines. Together, these data argue that (1) plasma is a more sensitive matrix for detecting changes in certain low-abundance cytokines; (2) calculation of concentrations in serum or plasma matrices is inherently inaccurate; and (3) dilution of samples should not be assumed to be linear, i.e., all comparisons need to be made among similarly diluted samples.

Breast Cancer Cell Colonization of the Human Bone Marrow Adipose Tissue Niche

BACKGROUND/OBJECTIVES: Bone is a preferred site of breast cancer metastasis, suggesting the presence of tissue-specific features that attract and promote the outgrowth of breast cancer cells. We sought to identify parameters of human bone tissue associated with breast cancer cell osteotropism and colonization in the metastatic niche. METHODS: Migration and colonization patterns of MDA-MB-231-fLuc-EGFP (luciferase-enhanced green fluorescence protein) and MCF-7-fLuc-EGFP breast cancer cells were studied in co-culture with cancellous bone tissue fragments isolated from 14 hip arthroplasties. Breast cancer cell migration into tissues and toward tissue-conditioned medium was measured in Transwell migration chambers using bioluminescence imaging and analyzed as a function of secreted factors measured by multiplex immunoassay. Patterns of breast cancer cell colonization were evaluated with fluorescence microscopy and immunohistochemistry. RESULTS: Enhanced MDA-MB-231-fLuc-EGFP breast cancer cell migration to bone-conditioned versus control medium was observed in 12/14 specimens (P = .0014) and correlated significantly with increasing levels of the adipokines/cytokines leptin (P = .006) and IL-1β (P = .001) in univariate and multivariate regression analyses. Fluorescence microscopy and immunohistochemistry of fragments underscored the extreme adiposity of adult human bone tissues and revealed extensive breast cancer cell colonization within the marrow adipose tissue compartment. CONCLUSIONS: Our results show that breast cancer cells migrate to human bone tissue-conditioned medium in association with increasing levels of leptin and IL-1β, and colonize the bone marrow adipose tissue compartment of cultured fragments. Bone marrow adipose tissue and its molecular signals may be important but understudied components of the breast cancer metastatic niche.

Cytokine signature associated with disease severity in chronic fatigue syndrome patients

Significance Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) devastates the lives of millions of people and has remained a mystery illness despite decades of research. It has long been suspected that inflammation is central to its pathogenesis. Although only two cytokines were found to be different (TGF-β higher and resistin lower) in ME/CFS patients compared with controls, 17 cytokines correlated with ME/CFS severity. Thirteen of these cytokines are proinflammatory and may contribute to many of the symptoms these patients experience for several years. Only CXCL9 (MIG) inversely correlated with fatigue duration. Although some signs of inflammation have been reported previously in patients with myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS), the data are limited and contradictory. High-throughput methods now allow us to interrogate the human immune system for multiple markers of inflammation at a scale that was not previously possible. To determine whether a signature of serum cytokines could be associated with ME/CFS and correlated with disease severity and fatigue duration, cytokines of 192 ME/CFS patients and 392 healthy controls were measured using a 51-multiplex array on a Luminex system. Each cytokine’s preprocessed data were regressed on ME/CFS severity plus covariates for age, sex, race, and an assay property of newly discovered importance: nonspecific binding. On average, TGF-β was elevated (P = 0.0052) and resistin was lower (P = 0.0052) in patients compared with controls. Seventeen cytokines had a statistically significant upward linear trend that correlated with ME/CFS severity: CCL11 (Eotaxin-1), CXCL1 (GROα), CXCL10 (IP-10), IFN-γ, IL-4, IL-5, IL-7, IL-12p70, IL-13, IL-17F, leptin, G-CSF, GM-CSF, LIF, NGF, SCF, and TGF-α. Of the 17 cytokines that correlated with severity, 13 are proinflammatory, likely contributing to many of the symptoms experienced by patients and establishing a strong immune system component of the disease. Only CXCL9 (MIG) inversely correlated with fatigue duration.

Correlates of Immunity to Influenza as Determined by Challenge of Children with Live, Attenuated Influenza Vaccine

Background. The efficacy of live, attenuated live attenuated influenza vaccine(LAIV) and inactivated influenza vaccine(IIV) is poorly explained by either single or composite immune responses to vaccination. Protective biomarkers were therefore studied in response to LAIV or IIV followed by LAIV challenge in children. Methods. Serum and mucosal responses to LAIV or IIV were analyzed using immunologic assays to assess both quantitative and functional responses. Cytokines and chemokines were measured in nasal washes collected before vaccination, on days 2, 4, and 7 after initial LAIV, and again after LAIV challenge using a 63-multiplex Luminex panel. Results. Patterns of immunity induced by LAIV and IIV were significantly different. Serum responses induced by IIV, including hemagglutination inhibition, did not correlate with detection or quantitation of LAIV on subsequent challenge. Modalities that induced sterilizing immunity seen after LAIV challenge could not be defined by any measurements of mucosal or serum antibodies induced by the initial LAIV immunization. No single cytokine or chemokine was predictive of protection. Conclusions. The mechanism of protective immunity observed after LAIV could not be defined, and traditional measurements of immunity to IIV did not correlate with protection against an LAIV challenge.

Multiple cytokine profile in plasma and amniotic fluid in a mouse model of pre-term labor.

Problem  The rate of pre‐term birth in the United States continues to rise despite several interventions. Induction of pro‐inflammatory cytokines and chemokines has been implicated in the activation of the cascade of events resulting in pre‐term labor. To date, no comprehensive panel of the cytokine profile in PTL has been published.

TOWARDS A CYTOKINE-CELL INTERACTION KNOWLEDGEBASE OF THE ADAPTIVE IMMUNE SYSTEM

The immune system of higher organisms is, by any standard, complex. To date, using reductionist techniques, immunologists have elucidated many of the basic principles of how the immune system functions, yet our understanding is still far from complete. In an era of high throughput measurements, it is already clear that the scientific knowledge we have accumulated has itself grown larger than our ability to cope with it, and thus it is increasingly important to develop bioinformatics tools with which to navigate the complexity of the information that is available to us. Here, we describe ImmuneXpresso, an information extraction system, tailored for parsing the primary literature of immunology and relating it to experimental data. The immune system is very much dependent on the interactions of various white blood cells with each other, either in synaptic contacts, at a distance using cytokines or chemokines, or both. Therefore, as a first approximation, we used ImmuneXpresso to create a literature derived network of interactions between cells and cytokines. Integration of cell-specific gene expression data facilitates cross-validation of cytokine mediated cell-cell interactions and suggests novel interactions. We evaluate the performance of our automatically generated multi-scale model against existing manually curated data, and show how this system can be used to guide experimentalists in interpreting multi-scale, experimental data. Our methodology is scalable and can be generalized to other systems.

ORIGINAL ARTICLE: Multiple Cytokine Profile in Plasma and Amniotic Fluid in a Mouse Model of Pre-Term Labor

Problem  The rate of pre‐term birth in the United States continues to rise despite several interventions. Induction of pro‐inflammatory cytokines and chemokines has been implicated in the activation of the cascade of events resulting in pre‐term labor. To date, no comprehensive panel of the cytokine profile in PTL has been published.

Cytokines profile in hypertensive patients with left ventricular remodeling and dysfunction.

There is strong evidence that inflammatory mediators play a key role in the progression to heart failure in patients with systemic hypertension (HTN). The present study aimed to identify a set of cytokines that are associated with early left ventricular (LV) remodeling and dysfunction as captured by echocardiography in patients with HTN in a cross-sectional case-control study nested within the FLEMish study on ENvironment, Genes and Health Outcome. We identified three groups of participants from the cohort: normotensive subjects (normotension; n = 30), HTN with normal LV structure and function (HTN [LV-]; n = 30), and HTN with evidence of adverse LV remodeling (HTN [LV+]; n = 50). We measured cytokines using a 63-plex Luminex platform. Using partial least squares-discriminant analysis, we constructed three latent variables from the measured cytokines that explained 35%-45% of the variance between groups. We identified five common cytokines (interleukin 18, monokine induced by gamma interferon, hepatocyte growth factor, epithelial neutrophil-activating peptide 78, and vascular endothelial growth factor D) with a stable signal which had a major impact on the construction of the latent variables. Among these cytokines, after adjustment for confounders, interleukin 18 remained significantly different between HTN participants with and without LV involvement (P = .02). Moreover, granulocyte-macrophage colony-stimulating factor and leptin showed a consistent upward trend in all HTN patients compared with normotensive subjects. In conclusion, in HTN patients with LV remodeling or/and dysfunction, we identified a set of cytokines strongly associated with LV maladaptation. We also found a distinct profile of inflammatory biomarkers that characterize HTN.

Circulating Biomarkers to Identify Responders in Cardiac Cell therapy

Bone marrow mononuclear cell (BM-MNC) therapy in ST-elevation acute myocardial infarction (STEMI) has no biological inclusion criteria. Here, we analyzed 63 biomarkers and cytokines in baseline plasma samples from 77 STEMI patients treated with BM-MNCs in the TIME and Late-TIME trials as well as 61 STEMI patients treated with placebo. Response to cell therapy was defined by changes in left ventricular ejection fraction, systolic/diastolic volumes, and wall motion indexes. We investigated the clinical value of circulating proteins in outcome prediction using significance testing, partial least squares discriminant analysis, and receiver operating characteristic (ROC) analysis. Responders had higher biomarker levels (76–94% elevated) than non-responders. Several biomarkers had values that differed significantly (P < 0.05) between responders and non-responders including stem cell factor, platelet-derived growth factor, and interleukin-15. We then used these lead candidates for ROC analysis and found multiple biomarkers with values areas under the curve >0.70 including interleukin 15. These biomarkers were not involved in the placebo-treated subjects suggesting that they may have predictive power. We conclude that plasma profiling after STEMI may help identify patients with a greater likelihood of response to cell-based treatment. Prospective trials are needed to assess the predictive value of the circulating biomarkers.

The Stanford Data Miner: a novel approach for integrating and exploring heterogeneous immunological data

BackgroundSystems-level approaches are increasingly common in both murine and human translational studies. These approaches employ multiple high information content assays. As a result, there is a need for tools to integrate heterogeneous types of laboratory and clinical/demographic data, and to allow the exploration of that data by aggregating and/or segregating results based on particular variables (e.g., mean cytokine levels by age and gender).MethodsHere we describe the application of standard data warehousing tools to create a novel environment for user-driven upload, integration, and exploration of heterogeneous data. The system presented here currently supports flow cytometry and immunoassays performed in the Stanford Human Immune Monitoring Center, but could be applied more generally.ResultsUsers upload assay results contained in platform-specific spreadsheets of a defined format, and clinical and demographic data in spreadsheets of flexible format. Users then map sample IDs to connect the assay results with the metadata. An OLAP (on-line analytical processing) data exploration interface allows filtering and display of various dimensions (e.g., Luminex analytes in rows, treatment group in columns, filtered on a particular study). Statistics such as mean, median, and N can be displayed. The views can be expanded or contracted to aggregate or segregate data at various levels. Individual-level data is accessible with a single click. The result is a user-driven system that permits data integration and exploration in a variety of settings. We show how the system can be used to find gender-specific differences in serum cytokine levels, and compare them across experiments and assay types.ConclusionsWe have used the tools and techniques of data warehousing, including open-source business intelligence software, to support investigator-driven data integration and mining of diverse immunological data.