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Metabolites produced in the human gut are known modulators of host immunity. However, large-scale identification of metabolite–host receptor interactions remains a daunting challenge. Here, we employed computational approaches to identify 983 potential metabolite–target interactions using the Inflammatory Bowel Disease (IBD) cohort dataset of the Human Microbiome Project 2 (HMP2). Using a consensus of multiple machine learning methods, we ranked metabolites based on importance to IBD, followed by virtual ligand-based screening to identify possible human targets and adding evidence from compound assay, differential gene expression, pathway enrichment, and genome-wide association studies. We confirmed known metabolite–target pairs such as nicotinic acid–GPR109a or linoleoyl ethanolamide–GPR119 and inferred interactions of interest including oleanolic acid–GABRG2 and alpha-CEHC–THRB. Eleven metabolites were tested for bioactivity in vitro using human primary cell-types. By expanding the universe of possible microbial metabolite–host protein interactions, we provide multiple drug targets for potential immune-therapies., Using computational approaches, Nuzzo et al. identify 983 potential metabolite–human target interactions from the Inflammatory Bowel Disease (IBD) cohort dataset of the Human Microbiome Project 2 (HMP2) and public databases. These predicted interactions can further the understanding of host–microbiome interactions and assist in drug discovery for IBD and other diseases.

Phenotypic drug discovery (PDD) uses biological systems directly for new drug screening. While PDD has proved effective in the discovery of drugs with novel mechanisms, for broader adoption, key challenges need resolution: progression of poorly qualified leads and overloaded pipelines due to lack of effective tools to process and prioritize hits; and advancement of leads with undesirable mechanisms that fail at more expensive stages of discovery. Here I discuss how human-based phenotypic platforms are being applied throughout the discovery process for hit triage and prioritization, for elimination of hits with unsuitable mechanisms, and for supporting clinical strategies through pathway-based decision frameworks. Harnessing the data generated in these platforms can also fuel a deeper understanding of drug efficacy and toxicity mechanisms. As these approaches increase in use, they will gain in power for driving better decisions, generating better leads faster and in turn promoting greater adoption of PDD.

Phenotypic assays using in vitro cell cultures to forecast compound effects in people are transforming pharmaceutical research and contribute to alternative methods for chemical safety testing. How these assays are validated for human disease relevance is a critical factor for developing more predictive assays. Chemical biology, using drugs as well as target-selective chemical probes, is a direct and efficient approach for establishing disease relevance. Chemical probes can connect information across assays and associate targets to clinical effects. When applied at scale, phenotypic chemical biology advances our understanding of drug and toxicity mechanisms enabling construction of disease outcome pathways. To improve the physiological relevance of phenotypic assays, standardized testing of a curated set of phenotypic pathway probes can provide a higher level of validation for phenotypic assay best practices.

Excipients, considered “inactive ingredients,” are a major component of formulated drugs and play key roles in their pharmacokinetics. Despite their pervasiveness, whether they are active on any targets has not been systematically explored. We computed the likelihood that approved excipients would bind to molecular targets. Testing in vitro revealed 25 excipient activities, ranging from low-nanomolar to high-micromolar concentration. Another 109 activities were identified by testing against clinical safety targets. In cellular models, five excipients had fingerprints predictive of system-level toxicity. Exposures of seven excipients were investigated, and in certain populations, two of these may reach levels of in vitro target potency, including brain and gut exposure of thimerosal and its major metabolite, which had dopamine D3 receptor dissociation constant Kd values of 320 and 210 nM, respectively. Although most excipients deserve their status as inert, many approved excipients may directly modulate physiologically relevant targets.

Nonclinical tests are considered crucial for understanding the safety of investigational medicines. However, the effective translation from nonclinical to human application is limited and must be improved. Drug development stakeholders are working to advance human-based in vitro and in silico methods that may be more predictive of human efficacy and safety in vivo because they enable scientists to model the direct interaction of drugs with human cells, tissues, and biological processes. Here, we recommend test-neutral regulations; increased funding for development and integration of human-based approaches; support for existing initiatives that advance human-based approaches; evaluation of new approaches using human data; establishment of guidelines for procuring human cells and tissues for research; and additional training and educational opportunities in human-based approaches.

Background The BioMAP® platform is a complex human primary cell-based system for modelling tissue and disease states that is used to characterise drug activities based on the analyses of 148 clinically relevant biomarker readouts. Objectives To confirm that the BioMAP phenotypic signatures of two originator etanercept (ETN) samples remained comparable over time. Methods Two different ETN samples (ETN_1 and ETN_2) were independently profiled with a 5 year interval across a panel of 12 disease-relevant systems (3C and 4 hour [endothelial inflammation]; LPS [monocyte activation]; SAg [T cell activation]; BT [B cell activation]; BF4T and BE3C [epithelial inflammation]; CASM3C [vascular inflammation]; HDF3CGF, KF3CT, and MyoF [tissue remodelling, fibrosis]; lMphg [macrophage activation]). BioMAP systems consist of human primary cells or cocultures from healthy donors cultured in the presence of cytokines and growth factors. Protein levels, measured using immune-based methods or functional assays for cell viability and proliferation, were used to generate a BioMAP activity profile for each sample. ETN activities were annotated if they differed from the vehicle control and had an effect size >20%. The profiles of the 2 samples were compared using Pearson’s correlation. Results BioMAP phenotypic profiling of ETN_1 versus ETN_2 samples at 10 µg/mL revealed similar signatures across 148 biomarkers in 12 disease-relevant systems. The Pearson’s correlation coefficient was 0.781, which is above the determined threshold for mechanistic similarity (r≥0.7). Key efficacy-related anti-inflammatory and immunomodulatory activities were commonly inhibited in multiple systems including tumour necrosis factor alpha (LPS and BT), interleukin (IL)−2 (BT), vascular cell adhesion molecule 1 (MyoF and lMphg), IL-8 (SAg and MyoF), and E-Selectin (SAg and lMphg). The profiles of ETN samples at 1 µg/mL in the SAg system modelling T cell activation responses also revealed statistically significant similarity in signatures (p Conclusions The BioMAP phenotypic signatures of the ETN_1 and ETN_2 samples profiled in independent experiments using different primary cell pools remained comparable, which was consistent with conserved ETN mechanisms of action. The BioMAP platform represents a useful orthogonal approach for assessing ETN activity. Disclosure of Interest A. O’Mahony Employee of: Eurofins DiscoverX, E. L. Berg Employee of: Eurofins DiscoverX, H. Jones Shareholder of: Pfizer, Employee of: Pfizer, B. Fitzpatrick Shareholder of: Pfizer, Employee of: Pfizer, B. Hassett Shareholder of: Pfizer, Employee of: Pfizer, S. Vicik Shareholder of: Pfizer, Employee of: Pfizer, L. Marshall Shareholder of: Pfizer, Employee of: Pfizer, K. Roshak: None declared, E. Choy Grant/research support from: Novimmune, Pfizer, Roche, UCB, Consultant for: Abbott Laboratories, Amgen, Biogen, BMS, Celgene, Chugai Pharma, Eli Lilly, GSK, Hospira, Janssen, MedImmune, Napp, Novimmune, Novartis, Pfizer, Regeneron, Roche, R-Pharm, Sanofi

The ToxCast program has generated in vitro screening data on over a thousand chemicals to assess potential disruption of important biological processes and assist in hazard identification and chemical testing prioritization. Few results have been reported for complex mixtures. To extend these ToxCast efforts to mixtures, we tested extracts from 30 organically grown fruits and vegetables in concentration-response in the BioMAP® assays. BioMAP systems use human primary cells primed with endogenous pathway activators to identify phenotypic perturbations related to proliferation, inflammation, immunomodulation, and tissue remodeling. Clustering of bioactivity profiles revealed separation of these produce extracts and ToxCast chemicals. Produce extracts elicited 87 assay endpoint responses per item compared to 20 per item for ToxCast chemicals. On a molar basis, the produce extracts were 10 to 50-fold less potent and when constrained to the maximum testing concentration of the ToxCast chemicals, the produce extracts did not show activity in as many assay endpoints. Using intake adjusted measures of dose, the bioactivity potential was higher for produce extracts than for agrichemicals, as expected based on the comparatively small amounts of agrichemical residues present on conventionally grown produce. The evaluation of BioMAP readouts and the dose responses for produce extracts showed qualitative and quantitative differences from results with single chemicals, highlighting challenges in the interpretation of bioactivity data and dose-response from complex mixtures.

Here we describe a chemical biology approach for elucidating potential toxicity mechanisms for thrombosis-related side effects. This work takes advantage of a large chemical biology data set comprising the effects of known, well-characterized reference agents on the cell surface levels of tissue factor (TF) in a primary human endothelial cell-based model of vascular inflammation, the BioMAP® 3C system. In previous work with the Environmental Protection Agency (EPA) for the ToxCast™ program, aryl hydrocarbon receptor (AhR) agonists and estrogen receptor (ER) antagonists were found to share an usual activity, that of increasing TF levels in this system. Since human exposure to compounds in both chemical classes is associated with increased incidence of thrombosis-related side effects, we expanded this analysis with a large number of well-characterized reference compounds in order to better understand the underlying mechanisms. As a result, mechanisms for increasing (AhR, histamine H1 receptor, histone deacetylase or HDAC, hsp90, nuclear factor kappa B or NFκB, MEK, oncostatin M receptor, Jak kinase, and p38 MAPK) and decreasing (vacuolar ATPase or V-ATPase) and mTOR) TF expression levels were uncovered. These data identify the nutrient, lipid, bacterial, and hypoxia sensing functions of autophagy as potential key regulatory points controlling cell surface TF levels in endothelial cells and support the mechanistic hypothesis that these functions are associated with thrombosis-related side effects in vivo.

Cutaneous reactions represent one of the most common adverse drug effects observed in clinical trials leading to substantial compound attrition. Three negative allosteric modulators (NAMs) of metabotropic glutamate receptors (mGluRs), which represent an important target for neurological diseases, developed by Pfizer, were recently failed in preclinical development due to delayed type IV skin hypersensitivity observed in non-human primates (NHPs). Here we employed large-scale phenotypic profiling in standardized panels of human primary cell/co-culture systems to characterize the skin toxicity mechanism(s) of mGluR5 NAMs from two different series. Investigation of a database of chemicals tested in these systems and transcriptional profiling suggested that the mechanism of toxicity may involve modulation of nuclear receptor targets RAR/RXR, and/or VDR with AhR antagonism. The studies reported here demonstrate how phenotypic profiling of preclinical drug candidates using human primary cells can provide insights into the mechanisms of toxicity and inform early drug discovery and development campaigns.

Introduction Unexpected drug activities account for many of the failures of new chemical entities in clinical trials. These activities can be target-dependent, resulting from feedback mechanisms downstream of the primary target, or they can occur as a result of unanticipated secondary target(s). Methods that would provide rapid and efficient characterization of compounds with respect to a broad range of biological pathways and mechanisms relevant to human disease have the potential to improve preclinical and clinical success rates. Methods BioMAP assays containing primary human cells (endothelial cells and co-cultures with peripheral blood leukocytes) were stimulated in complex formats (specific combinations of inflammatory mediators) for 24 h in the presence or absence of test agents (drugs, experimental compounds, etc.). The levels of selected protein readouts (adhesion receptors, cytokines, enzymes, etc.) were measured and activity profiles (normalized data sets comprising BioMAP profiles) were generated for each test agent. The resulting profiles were compared by statistical methods to identify similarities and mechanistic insights. Results Compounds with known mechanisms including inhibitors of histamine H1 receptor, angiotensin converting enzyme, IκB kinase-2, β2 adrenergic receptor and others were shown to generate reproducible and distinguishable BioMAP activity profiles. Similarities were observed between compounds targeting components within the same signal transduction pathway (e.g. NFκB), and also between compounds that share secondary targets (e.g. ibuprofen and FMOC-L-leucine, a PPARγ agonist). Discussion Complex primary cell-based assays can be applied for detecting and distinguishing unexpected activities that may be of relevance to drug action in vivo. The ability to rapidly test compounds prior to animal or clinical studies may reduce the number of compounds that unexpectedly fail in preclinical or clinical studies.

The hope of the rapid translation of 'genes to drugs' has foundered on the reality that disease biology is complex, and that drug development must be driven by insights into biological responses. Systems biology aims to describe and to understand the operation of complex biological systems and ultimately to develop predictive models of human disease. Although meaningful molecular level models of human cell and tissue function are a distant goal, systems biology efforts are already influencing drug discovery. Large-scale gene, protein and metabolite measurements ('omics') dramatically accelerate hypothesis generation and testing in disease models. Computer simulations integrating knowledge of organ and system-level responses help prioritize targets and design clinical trials. Automation of complex primary human cell-based assay systems designed to capture emergent properties can now integrate a broad range of disease-relevant human biology into the drug discovery process, informing target and compound validation, lead optimization, and clinical indication selection. These systems biology approaches promise to improve decision making in pharmaceutical development.

Addressing the safety aspects of drugs and environmental chemicals has historically been undertaken through animal testing. However, the quantity of chemicals in need of assessment and the challenges of species extrapolation require the development of alternative approaches. Our approach, the US Environmental Protection Agency's ToxCast program, utilizes a large suite of in vitro and model organism assays to interrogate important chemical libraries and computationally analyze bioactivity profiles. Here we evaluated one component of the ToxCast program, the use of primary human cell systems, by screening for chemicals that disrupt physiologically important pathways. Chemical-response signatures for 87 endpoints covering molecular functions relevant to toxic and therapeutic pathways were generated in eight cell systems for 641 environmental chemicals and 135 reference pharmaceuticals and failed drugs. Computational clustering of the profiling data provided insights into the polypharmacology and potential off-target effects for many chemicals that have limited or no toxicity information. The endpoints measured can be closely linked to in vivo outcomes, such as the upregulation of tissue factor in endothelial cell systems by compounds linked to the risk of thrombosis in vivo. Our results demonstrate that assaying complex biological pathways in primary human cells can identify potential chemical targets, toxicological liabilities and mechanisms useful for elucidating adverse outcome pathways.

Objective To determine the role of the endothelial cell adhesion molecules E- and P-selectin in the development and severity of adjuvant-induced arthritis (AIA) in the rat. Methods Lewis rats were immunized subcutaneously with Mycobacterium butyricum (Mb), and blocking monoclonal antibodies (mAb) to rat E- and P-selectin were administered. Clinical score, radiolabeled (51Cr and 111In) blood polymorphonuclear leukocyte (PMN) and monocyte migration to joints, and histologic features were monitored. Results When mAb treatment was started on day 5 postimmunization with Mb (preclinical stage), development of AIA was significantly (P < 0.01) inhibited by mAb to E- but not to P-selectin (mean score on day 14 control 10.2, anti-E 2.8, anti-P 9.1). This was associated with markedly decreased migration (by 66–94%) of PMN and monocytes to arthritic joints and diminished cartilage degradation. When treatment was delayed until animals showed signs of arthritis (day 10 postimmunization), only a marginal and variable effect was observed as compared with blockade during the preclinical (day 5) stage. E-selectin blockade on day 5 and day 7 postimmunization resulted in inhibition of antigen-dependent T cell–mediated inflammation, since it decreased T cell migration to sites of dermal-delayed hypersensitivity induced by Mb without affecting migration to concanavalin A or cytokines. The proliferative response of T cells to Mb in vitro was not altered. Conclusion E-selectin plays an important role early in the development of AIA. This adhesion molecule may contribute to the migration of antigen-reactive T cells to peripheral tissues, including the joints where T cells initiate the arthritis.

Background —After activation, platelets adhere to neutrophils via P-selectin and β 2 -integrin. The molecular mechanisms and adhesion events in whole blood exposed to venous levels of hydrodynamic shear in the absence of exogenous activation remain unknown. Methods and Results —Whole blood was sheared at ≈100 s −1 . The kinetics of neutrophil-platelet adhesion and neutrophil aggregation were measured in real time by flow cytometry. P-selectin was upregulated to the platelet surface in response to shear and was the primary factor mediating neutrophil-platelet adhesion. The extent of neutrophil aggregation increased linearly with platelet adhesion to neutrophils. Blocking either P-selectin, its glycoprotein ligand PSGL-1, or both simultaneously by preincubation with a monoclonal antibody resulted in equivalent inhibition of neutrophil-platelet adhesion (≈30%) and neutrophil aggregation (≈70%). The residual amount of neutrophil adhesion was blocked with anti-CD11b/CD18. Treatment of blood with prostacyclin analogue ZK36374, which raises cAMP levels in platelets, blocked P-selectin upregulation and neutrophil aggregation to baseline. Complete abrogation of platelet-neutrophil adhesion required both ZK36374 and anti-CD18. Electron microscopic observations of fixed blood specimens revealed that platelets augmented neutrophil aggregation both by forming bridges between neutrophils and through contact-mediated activation. Conclusions —The results are consistent with a model in which venous levels of shear support platelet adherence to neutrophils via P-selectin binding PSGL-1. This interaction alone is sufficient to mediate neutrophil aggregation. Abrogation of platelet adhesion and aggregation requires blocking Mac-1 in addition to PSGL-1 or P-selectin. The described mechanisms are likely of key importance in the pathogenesis and progression of thrombotic disorders that are exacerbated by leukocyte-platelet aggregation.

Interactions between lymphocyte surface receptors and their ligands on vascular endothelial cells regulate the exit of lymphocytes from the circulation. Distinct subsets of mononuclear cells bind to high endothelial venules (HEVs) in different lymphoid organs to a different extent, but the molecular mechanisms behind this selectivity have remained poorly characterized. Here we show that vascular adhesion protein-1 (VAP-1) mediates subtype-specific binding of CD8-positive T cells and natural killer cells to human endothelium. VAP-1–dependent, oligosaccharide-dependent peripheral lymph node (PLN) HEV adhesion under shear was independent of L-selectin, P-selectin glycoprotein ligand 1, and α4 integrins, the known lymphocyte receptors involved in the initial recognition of endothelial cells. PLN HEV adhesion was also critically dependent on peripheral lymph node vascular addressins (PNAds), but lymphocyte L-selectin was absolutely required for PNAd binding. Most lymphocytes relied on both PNAd and VAP-1 in HEV binding. The overlapping function of L-selectin ligands and VAP-1 in PLN introduces a new control point into the lymphocyte extravasation process. Finally, intravital microscopy revealed that VAP-1 is involved in initial interactions between human lymphocytes and endothelial cells in inflamed rabbit mesenterial venules in vivo. In conclusion, VAP-1 is a novel contact-initiating ligand that discriminates between different subpopulations of mononuclear cells and is an appealing target for selective modulation of adhesion of CD8- and CD16-positive effector cells.

Innovation and new molecular entity production by the pharmaceutical industry has been below expectations. Surprisingly, more first-in-class small-molecule drugs approved by the U.S. Food and Drug Administration (FDA) between 1999 and 2008 were identified by functional phenotypic lead generation strategies reminiscent of pre-genomics pharmacology than contemporary molecular targeted strategies that encompass the vast majority of lead generation efforts. This observation, in conjunction with the difficulty in validating molecular targets for drug discovery, has diminished the impact of the "genomics revolution" and has led to a growing grassroots movement and now broader trend in pharma to reconsider the use of modern physiology-based or phenotypic drug discovery (PDD) strategies. This "From the Guest Editors" column provides an introduction and overview of the two-part special issues of Journal of Biomolecular Screening on PDD. Terminology and the business case for use of PDD are defined. Key issues such as assay performance, chemical optimization, target identification, and challenges to the organization and implementation of PDD are discussed. Possible solutions for these challenges and a new neoclassic vision for PDD that combines phenotypic and functional approaches with technology innovations resulting from the genomics-driven era of target-based drug discovery (TDD) are also described. Finally, an overview of the manuscripts in this special edition is provided.

Of the several families of adhesion receptors involved in leukocyte-endothelial cell interactions, only the selectins have been shown to initiate leukocyte interaction under physiologic shear; indeed, beta 2 (CD18) intergrins responsible for neutrophil arrest are unable to engage without prior selectin-mediated rolling. In contrast, alpha 4 (CD49d) integrins are shown here to initiate lymphocyte contract ("tethering") in vitro under shear and in the absence of a selectin contribution. The alpha 4 integrin ligands MAdCAM-1 and VCAM-1 support loose reversible interactions including rolling, as well as rapid sticking and arrest that is favored following integrin activation. Moreover, alpha 4 beta 7 mediates L-selectin (CD62L)-independent attachment of blood-borne lymphocytes to lamina propria venules in situ. Scanning electron microscopy of alpha 4 beta 7hi lymphoid cells reveals that, like L-selectin, alpha 4 beta 7 is highly concentrated on microvillous sites of initial cellular contact, whereas the beta 2 integrin LFA-1 is excluded from villi. Thus, alpha 4 but not beta 2 integrins can initiate leukocyte adhesion under flow, a capacity that may be in part a function of topographic presentation on microvilli.

E- and P-selectin are inflammation-induced cell adhesion molecules that mediate leukocyte-endothelial cell and leukocyte-platelet interactions. Monoclonal antibodies (MoAbs) specific for either E-selectin or P- selectin are protective in several animal models of inflammatory disease. To generate an MoAb with broader therapeutic potential, MoAbs that bind to both E- and P-selectin were generated by immunization of mice with mouse pre-B cell lines transfected with human E- and P- selectin. Interestingly, although the only selection criterion was the ability to bind both E- and P-selectin, all three antibodies obtained efficiently block both E- and P-selectin-mediated functions. The inhibited functions include neutrophil or HL-60 cell binding to tumor necrosis factor-alpha-activated human umbilical vein endothelial cells, E- or P-selectin transfectant cell lines, and platelet-HL-60 rosetting. These antibodies, EP-5C7, EP-2C9, and EP-1D8, recognize the same or overlapping epitope within the lectin domains of E- and P-selectin. The data suggest that functionally important epitopes of homologous proteins can be targeted by selecting for antibodies with reactivity toward both proteins. Furthermore, a potent blocking antibody specific for both E- and P-selectin may provide a more effective and broadly useful reagent for treating acute and potentially certain chronic inflammatory conditions.

The glycoprotein (GP) L-selectin initiates adhesive interactions between leukocytes and endothelial cells (EC). It functions as a lymphocyte-lectin homing receptor recognizing carbohydrate determinants of the peripheral lymph node addressing on high endothelial venules. It also mediates neutrophil rolling, the earliest interaction of neutrophils with acutely inflamed venules. Neutrophil L-selectin presents sialyl-LewisX (sLe(X)) as a ligand to P- and E-selectin in vitro, and we have proposed that this is a major mechanism of L- selectin-mediated rolling in vivo. In contrast, the contribution of neutrophil L-selectin as a receptor protein recognizing one (or more) ligand(s) on inflamed EC is unclear. To address this question, an sLe(X)-negative murine pre-B cell line, L1–2, that can neither bind vascular selectins nor roll in inflamed rabbit venules, was transfected with human L-selectin cDNA. L-selectin expression in stable transfectants was sufficient to confer significant rolling in vivo. Rolling was unaffected by neuraminidase treatment but completely blocked by anti-L-selectin monoclonal antibody (MoAb) DREG-56. Thus, L- selectin can initiate leukocyte interactions with EC determinants potentially through recognition of endothelial carbohydrates. In contrast, when human neutrophils were tested, rolling was reduced, but not abolished, by MoAb DREG-56. Likewise, treatment with neuraminidase or anti-sLe(X) MoAbs decreased, but did not abrogate, neutrophil rolling, consistent with residual EC recognition via L-selectin. Combination of MoAb DREG-56 and neuraminidase resulted in almost complete loss of rolling, as did removal of glycosylated L-selectin by chymotrypsin. Together with the demonstrable rolling of L-selectin transfectants, our results support the concept of a bidirectional interaction between L-selectin bearing sLe(X) on neutrophils and activated EC in vivo. These findings also suggest that L-selectin may mediate rolling of lymphocytes that lack carbohydrate ligands for E- or P-selectin, although probably less efficiently than through bidirectional recognition.

The complexity of human biology has made prediction of health effects as a consequence of exposure to environmental chemicals especially challenging. Complex cell systems, such as the Biologically Multiplexed Activity Profiling (BioMAP) primary, human, cell-based disease models, leverage cellular regulatory networks to detect and distinguish chemicals with a broad range of target mechanisms and biological processes relevant to human toxicity. Here the authors use the BioMAP human cell systems to characterize effects relevant to human tissue and inflammatory disease biology following exposure to the 320 environmental chemicals in the Environmental Protection Agency's (EPA's) ToxCast phase I library. The ToxCast chemicals were assayed at 4 concentrations in 8 BioMAP cell systems, with a total of 87 assay endpoints resulting in more than 100,000 data points. Within the context of the BioMAP database, ToxCast compounds could be classified based on their ability to cause overt cytotoxicity in primary human cell types or according to toxicity mechanism class derived from comparisons to activity profiles of BioMAP reference compounds. ToxCast chemicals with similarity to inducers of mitochondrial dysfunction, cAMP elevators, inhibitors of tubulin function, inducers of endoplasmic reticulum stress, or NFkappaB pathway inhibitors were identified based on this BioMAP analysis. This data set is being combined with additional ToxCast data sets for development of predictive toxicity models at the EPA.

Introduction The ability to predict the health effects resulting from drug or chemical exposure has been challenging due to the complexity of human biology. Approaches that detect and discriminate a broad range of mechanisms in testing formats that are predictive and yet cost-effective are needed. Methods Here, we evaluated the performance of BioMAP systems, primary human cell-based disease models, as a platform for characterization of chemical toxicity mechanisms. For this we tested a set of compounds with known or well-studied mechanisms in a panel of 8 BioMAP assays relevant to human respiratory, skin, immune and vascular exposure sites. Results We evaluated the ability to detect and distinguish compounds based on mechanisms of action, comparing the BioMAP activity profiles generated in a reduced sample number format to reference database profiles derived from multiple experiments. We also studied the role of BioMAP assay panel size and concentration effects, both of which were found to contribute to the ability to discriminate chemicals and mechanisms. Discussion Compounds with diverse mechanisms, including modulators of the NFκB pathway, microtubule function and mitochondrial activity, could be discriminated and classified into target and pathway mechanisms in both assay formats. Certain inhibitors of mitochondrial function, such as rotenone and sodium azide, but not others, were classified with inducers of endoplasmic reticulum stress, providing insight into the toxicity mechanisms of these agents. This method may have utility in classifying novel agents with unknown modes of action according to their effects on toxicity pathways.

The specificity of endothelial cell leukocyte adhesion molecule-1, ELAM-1, for binding to a panel of carbohydrate structures was determined by a sensitive cell binding assay with immobilized synthetic glycoconjugates. ELAM-1 cDNA transfectants were found to bind Sialyl Lea (sialylated lacto-N-fucopentaose II) or sialylated Lewis a antigen (NeuAc alpha 2-3Gal beta 1-3(Fuc alpha 1-4)GlcNAc), as well as or slightly better than Sialyl Lex (sialylated lacto-N-fucopentaose III) or sialylated Lewis X antigen (NeuAc alpha 2-3 Gal beta 1-4(Fuc alpha 1-3)GlcNAc). A monoclonal antibody, HECA-452, which has been identified recently as recognizing ELAM-1 ligands in addition to those containing Sialyl Lex, was also found to bind both Sialyl Lex and Sialyl Lea. Hard sphere exo-anomeric (HSEA) calculations were performed on these two hexasaccharides. The conformations indicate that Sialyl Lea and Sialyl Lex show a high degree of similarity in both the nonreducing and reducing termini. As Lea and Lex show much weaker reactivity, the determinants recognized by ELAM-1 and HECA-452 probably involve neuraminic acid and fucose residues which on one face of both Sialyl Lex and Sialyl Lea can be similarly positioned. The finding that Sialyl Lea is a potent ligand for ELAM-1 is important, as circulating Sialyl Lea and Sialyl Lex containing mucins which are elevated in the serum of many cancer patients may block leukocyte interactions with ELAM-1 and may contribute to the pathological immunodepression observed in these patients.

A skin-associated population of memory T lymphocytes, defined by expression of the cutaneous lymphocyte antigen (CLA), binds selectively and avidly to the vascular lectin endothelial cell-leukocyte adhesion molecule 1 (ELAM-1), an interaction that may be involved in targeting of CLA+ T cells to cutaneous sites of chronic inflammation. Here we present evidence that CLA itself is the (or a) lymphocyte homing receptor for ELAM-1. Antigen isolated with anti-CLA monoclonal antibody HECA-452 from human tonsillar lysates avidly binds ELAM-1 transfected mouse cells. Anti-CLA antibody blocks T lymphocyte binding to ELAM-1 transfectants. HECA-452 and ELAM-1 binding to lymphocytes or to isolated tonsillar HECA-452 antigen is abrogated by neuraminidase treatment implying a prominent role for sialic acid in CLA structure and function. The dominant form of CLA on T cells is immunologically distinct from the major neutrophil ELAM-1 ligand, the sialyl Lewis x (sLex) antigen (NeuAc alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc), which is absent, weakly expressed, or masked on T cells. However, neuraminidase treatment of CLA+ T cells, but not of CLA- T cells, reveals Lewis x (CD15) structures. In combination with the known requirement for terminal NeuAc alpha 2-3Gal and fucose residues attached to N-acetylglucosamine for ELAM-1 and HECA-452 binding, this finding suggests that CLA may comprise an additionally sialylated or otherwise modified form of sLex. The identification of a lymphocyte homing receptor for skin may permit novel approaches to the diagnosis and therapy of cutaneous and inflammatory disorders.

Now that the human genome has been sequenced, the challenge of assigning function to human genes has become acute. Existing approaches using microarrays or proteomics frequently generate very large volumes of data not directly related to biological function, making interpretation difficult. Here, we describe a technique for integrative systems biology in which: ( i ) primary cells are cultured under biologically meaningful conditions; ( ii ) a limited number of biologically meaningful readouts are measured; and ( iii ) the results obtained under several different conditions are combined for analysis. Studies of human endothelial cells overexpressing different signaling molecules under multiple inflammatory conditions show that this system can capture a remarkable range of functions by a relatively small number of simple measurements. In particular, measurement of seven different protein levels by ELISA under four different conditions is capable of reconstructing pathway associations of 25 different proteins representing four known signaling pathways, implicating additional participants in the NF-κBorRAS/mitogen-activated protein kinase pathways and defining additional interactions between these pathways.

Background A significant advance in rheumatoid arthritis therapy has been the development of biologics targeting proinflammatory cytokines. Biologics are commonly used with methotrexate (MTX) because clinical trials of anti–tumor necrosis factor alpha (TNFα) antibodies, such as adalimumab (ADA), have shown that combination therapy is more effective than monotherapy based, in part, on the assumption that MTX reduces the immunogenicity of biologics. However, combining MTX with the anti–interleukin-6 (IL-6) receptor antibody tocilizumab (TCZ) does not demonstrate the same level of incremental benefit over monotherapy. Given the known biologic effects of TNFα, IL-6, and MTX, we hypothesized that because IL-6 has more diverse effects on the immune system than TNFα, MTX may have fewer supplementary effects than TNFα inhibitors when added to TCZ. Objectives Using the BioMAP platform, we previously reported that TCZ, ADA, and MTX have distinct activity profiles. Here we used BioMAP systems to compare the profiles of combinations (ADA+MTX and TCZ+MTX) with the profiles of the individual agents (ADA or TCZ or MTX) to distinguish adjuvant effects. Methods BioMAP systems model disease biology in early-passage human primary cells, cultured alone or with different stimulus combinations, and have been used extensively to characterize compounds based on phenotypic signatures.1,2 BioMAP systems were used to generate profiles of TCZ, ADA, and MTX alone and of TCZ+MTX and ADA+MTX combinations. Agents were profiled at concentrations that would include their clinical Cmax ranges for the respective approved dosing regimen. Changes in protein-based and clinically relevant endpoints (biomarkers) and other cellular events (eg, proliferation, cell cytotoxicity) were evaluated to determine whether activities detected with the combinations were significantly different from the individual agents profiled in parallel. Results BioMAP profiling of ADA+MTX revealed more statistically significant different activities (p Conclusions These data show that TCZ has diverse effects on the immune response; the addition of MTX elicits few additional activities other than antiproliferative effects compared with TCZ alone. In contrast, ADA+MTX has statistically significantly more nonoverlapping effects on immune function, inflammation markers, and matrix-remodeling endpoints in primary human cell disease models than ADA or MTX alone. This finding supports our hypothesis that pharmacodynamic interactions between TCZ and MTX are less pronounced than those between MTX and ADA and may explain the clinical observation that TCZ is more effective than ADA as monotherapy.3 References Berg EL. Drug Discov Today. 2013; doi: 10.1016/j.drudis.2013.10.003. Berg EL et al. J Biomol Screen. 2013; doi: 10.1177/1087057113505324. Gabay C et al. Lancet. 2013;381:1541-1550. Disclosure of Interest : A. O9Mahony Shareholder of: DiscoveRx, Employee of: DiscoveRx, E. Berg Employee of: DiscoveRx, M. John Employee of: Roche, K. Ganeshalingam Shareholder of: Roche, Employee of: Roche, E. Choy Grant/research support: Abbott, Boehringer Ingelheim, Chelsea Therapeutics, Chugai Pharma, GSK, Jazz Pharmaceuticals, MSD, Novartis, Pierre Fabre Medicament, Roche, UCB, Consultant for: Abbott, Allergan, AstraZeneca, Boehringer Ingelheim, Chelsea Therapeutics, Chugai Pharma, Daiichi Sankyo, Eli Lilly, Ferring Pharmaceutical, GSK, ISIS, Jazz Pharmaceuticals, MedImmune, Merrimack Pharmaceutical, MSD, Novartis, Pfizer, Pierre Fabre Medicament, Roche, Schering Plough, Synovate, UCB, Speakers bureau: Abbott, Boehringer Ingelheim, Chugai Pharma, Eli Lilly, Jazz Pharmaceuticals, MSD, Novartis, Pfizer, Pierre Fabre Medicament, Roche, Scheering Plough, Synovate, UCB DOI 10.1136/annrheumdis-2014-eular.3386

Despite recent successful treatment of murine autoimmune disease with anti-IL-12 mAb, it has not yet been addressed whether anti-IL-12 mAb can also be effective in late stages of disease and whether it can provide lasting protection against recurrence, especially during continued presence of autoantigen. We used a newly developed psoriasis model in scid/scid mice, which allows easy tracking of pathogenic T cells, to show that when anti-IL-12 mAb is given for 2 wk (1 mg/wk) in the late stage of severe disease, inflammation is greatly reduced, as measured by ear thickness and histology (scores, 1.1 ± 0.1 vs 2.0 ± 0.4). Moreover, prolonged treatment (4 wk) of chronic psoriatic mice with high doses of mAb (1 mg/wk; prolonged active anti-inflammatory treatment (PAAIT)) results in the almost complete resolution of lesions (scores, 0.3 ± 0.1 vs 2.7 ± 0.2). Surprisingly, however, despite these significant treatment results, the psoriasis-like lesions return soon after the anti-IL-12 mAb treatment is discontinued. This rapid relapse of disease may be attributed to large populations of activated CD4+ T cells present in the lymph nodes of PAAIT animals still expressing an effector/memory phenotype (CD45RBlow, L-selectinlow). Upon stimulation in vitro such PAAIT lymph node cells secrete high amounts of IFN-γ (129 ng/ml); when transferred into naive scid/scid animals they are able to rapidly induce disease without costimulation. Our data indicates an alternative IL-12-independent pathway for pathogenic Th-1-like cells in vivo during the chronic phase of disease that allows these cells to persist and maintain their pathogenicity in the draining lymph tissue of the autoimmune site.

The chemokine receptor CCR5 is the major coreceptor for R5 human immunodeficiency virus type-1 strains. We mapped the epitope specificities of 18 CCR5 monoclonal antibodies (mAbs) to identify domains of CCR5 required for chemokine binding, gp120 binding, and for inducing conformational changes in Env that lead to membrane fusion. We identified mAbs that bound to N-terminal epitopes, extracellular loop 2 (ECL2) epitopes, and multidomain (MD) epitopes composed of more than one single extracellular domain. N-terminal mAbs recognized specific residues that span the first 13 amino acids of CCR5, while nearly all ECL2 mAbs recognized residues Tyr-184 to Phe-189. In addition, all MD epitopes involved ECL2, including at least residues Lys-171 and Glu-172. We found that ECL2-specific mAbs were more efficient than NH2- or MD-antibodies in blocking RANTES or MIP-1beta binding. By contrast, N-terminal mAbs blocked gp120-CCR5 binding more effectively than ECL2 mAbs. Surprisingly, ECL2 mAbs were more potent inhibitors of viral infection than N-terminal mAbs. Thus, the ability to block virus infection did not correlate with the ability to block gp120 binding. Together, these results imply that chemokines and Env bind to distinct but overlapping sites in CCR5, and suggest that the N-terminal domain of CCR5 is more important for gp120 binding while the extracellular loops are more important for inducing conformational changes in Env that lead to membrane fusion and virus infection. Measurements of individual antibody affinities coupled with kinetic analysis of equilibrium binding states also suggested that there are multiple conformational states of CCR5. A previously described mAb, 2D7, was unique in its ability to effectively block both chemokine and Env binding as well as coreceptor activity. 2D7 bound to a unique antigenic determinant in the first half of ECL2 and recognized a far greater proportion of cell surface CCR5 molecules than the other mAbs examined. Thus, the epitope recognized by 2D7 may represent a particularly attractive target for CCR5 antagonists.

Improper regulation of B cell responses leads to excessive production of antibodies and contributes to the development of autoimmune disease. T helper 17 (Th17) cells also drive the development of autoimmune disease, but the role of B cells in shaping Th17 cell-mediated immune responses, as well as the reciprocal regulation of B cell responses by IL-17 family cytokines, remains unclear. The aim of this study was to characterize the regulation of IL-17A and IL-17F in a model of T cell-dependent B cell activation. Stimulation of primary human B cell and peripheral blood mononuclear cell (BT) co-cultures with α-IgM and a non-mitogenic concentration of superantigens for three days promoted a Th17 cell response as evidenced by increased expression of Th17-related gene transcripts, including Il17f, Il21, Il22, and Il23r, in CD4 T cells, as well as the secretion of IL-17A and IL-17F protein. We tested the ability of 144 pharmacologic modulators representing 91 different targets or pathways to regulate IL-17A and IL-17F production in these stimulated BT co-cultures. IL-17A production was found to be preferentially sensitive to inhibition of the PI3K/mTOR pathway, while prostaglandin EP receptor agonists, including PGE2, increased IL-17A concentrations. In contrast, the production of IL-17F was inhibited by PGE2, but selectively increased by TLR2 and TLR5 agonists. These results indicate that IL-17A regulation is distinct from IL-17F in stimulated BT co-cultures and that this co-culture approach can be used to identify pathway mechanisms and novel agents that selectively inhibit production of IL-17A or IL-17F.

Background and Purpose E-selectin participates in leukocyte-endothelial adhesion and the inflammatory processes that follow focal cerebral ischemia and reperfusion. The temporal and topographical patterns of microvascular E-selectin presentation after experimental focal cerebral ischemia are relevant to microvascular reactivity to ischemia. Methods The upregulation and fate of E-selectin antigen during 2 hours of middle cerebral artery occlusion (n=4) and 3 hours of occlusion with reperfusion (1 hour, n=4; 4 hours, n=6; 24 hours, n=6) were evaluated in the nonhuman primate. E-selectin and E:P-selectin immunoreactivities were semiquantitated with the use of computerized light microscopy video imaging and laser confocal microscopy. Results Three patterns of microvascular E-selectin expression, defined by the antibody E-1E4, were confirmed by complete elimination of E-1E4 binding after incubation with soluble recombinant human E-selectin: (1) Low immunoperoxidase intensity was observed in ischemic microvessels at 2 hours of occlusion extending to 4 hours of reperfusion (E-selectin/laminin=0.32±0.10). (2) A significant fraction of ischemic microvessels displayed high-intensity E-selectin signal by 24 hours of reperfusion (0.61±0.17) compared with control and nonischemic tissues (2 P P =.0005). The latter were further confirmed by an E:P-selectin immunoprobe. Conclusions E-selectin antigen is distinctively and significantly upregulated in nonhuman primate brain after focal ischemia and reperfusion. The late appearance of E-selectin in nonischemic cerebral tissues suggests stimulation by transferable factors generated during brain injury.

Few markers specific for mouse endothelium exist. We describe here one such marker, MECA-32, a monoclonal antibody which shows high specificity for mouse endothelium in both embryonic and mature tissues. The MECA-32 antigen has a M(r) of 50-55 x 10(3) under reducing conditions and M(r) of 100-120 x 10(3) under nonreducing conditions. It is expressed on most endothelial cells in the embryonic and in the adult mouse, with the exception of the brain, skeletal, and cardiac muscle, where it has a more restricted distribution. In skeletal and cardiac muscle only small arterioles and venules express the MECA-32 antigen, while in the brain its expression is negatively correlated with the differentiation of the vasculature to form the blood brain barrier. Interestingly, during embryonic development the antigen occurs on the brain vasculature up to day 16 of gestation (E16), whereupon it disappears. The embryonic brain is an avascular organ anlage which is vascularized by ingrowth of external blood vessels. Differentiation of the vasculature to form the blood brain barrier occurs at approximately E16 in the mouse. This differentiation correlates with the downregulation of MECA-32 antigen expression. Between E12 and E16 MECA-32 detects most endothelial cell surfaces of the blood vessels in the brain. No MECA-32 antigen is found in the brain at E17 or any later stage of development with the exception of the vasculature of the circumventricular organs. The results suggest that MECA-32 antigen expression is temporally and spatially correlated with the development of the blood brain barrier.

It is now widely accepted that chronic inflammatory events are critical contributors to both the development and progression of cancer. While the complex interrelationship between immune events and their effects on the neoplastic process remain to be defined, targeting inflammation has now become a priority of drug discovery in oncology. A major factor propelling this strategy is that both cancer cells and immune cells share many common signaling pathways that can contribute to the disease promoting microenvironment of the tumor. Drugs that target these pathways now make up a significant portion of pharmaceutical pipelines but how these compounds affect cancer cells, normal tissues and immune processes remains poorly understood. In order to develop more effective therapeutic strategies it is imperative that we understand the biological impact of compounds that target key signaling intermediates on normal and immune cells as well as neoplastic cells. BioSeek has pioneered the development of in vitro human cell-based systems (BioMAP® Systems) that recapitulate the complex signals and phenotypic responses of inflamed tissues in vivo. In addition to providing broad coverage of inflammatory targets, BioMAP® Systems model modulation of many signaling pathways relevant to cancer. Characterization of pathway-associated kinase inhibitors, including JAK, PI3K, p38MAPK, ERK, JNK and NF-κB in BioMAP® Systems has revealed activities that differentially impact inflammatory, proliferative, angiogenic and tissue remodeling processes. Secondly, profiling of 1000s of off-patent compounds through BioMAP® Systems identified agents that selectively prevent the growth of smooth muscle cells and related cancers (leiomyosarcomas and rhabdomyosarcomas) with little impact on the activation of immune cells. BioMAP® Systems provide a highly useful platform to (1) distinguish anti-inflammatory and anti-proliferative effects of various kinase inhibitors and (2) identify different phenotypic outcomes of these agents that influence cancer progression and disease resolution. Together these data will help inform the selection of more effective therapeutic strategies to target different cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1093. doi:10.1158/1538-7445.AM2011-1093

The L-selectin, a cell surface C-type lectin, directs lymphocyte traffic to lymph nodes, and contributes to lymphocyte homing to Peyer's patches and to leukocyte interactions with inflamed venules. Here we report that the mucosal vascular addressin MAdCAM-1, a mucosal endothelial adhesion molecule with immunoglobulin- and mucin-like domains, is a facultative ligand for L-selectin. MAdCAM-1 isolated from mesenteric lymph nodes, but not from cultured endothelioma cells, bears N-glycanase-resistant sialic acid-containing carbohydrate which supports adhesion of L-selectin-transfected lymphoid cells under shear. Interacting lymphoid cells display a 'rolling' behaviour similar to the selectin-dependent rolling of neutrophils observed in inflamed venules. MAdCAM-1 is also a ligand for the lymphocyte integrin homing receptor for Peyer's patches, alpha 4 beta 7 (ref. 7), and may be uniquely adapted to support both selectin-mediated lymphocyte rolling and integrin-mediated adhesion and arrest in vivo.

The trafficking of lymphocytes from the blood and into lymphoid organs is controlled by tissue-selective lymphocyte interactions with specialized endothelial cells lining post capillary venules, in particular the high endothelial venules (HEV) found in lymphoid tissues and sites of chronic inflammation. Lymphocyte interactions with HEV are mediated in part by lymphocyte homing receptors and tissue-specific HEV determinants, the vascular addressins. A peripheral lymph node addressin (PNAd) has been detected immunohistologically in mouse and man by monoclonal antibody MECA-79, which inhibits lymphocyte homing to lymph nodes and lymphocyte binding to lymph node and tonsillar HEV. The human MECA-79 antigen, PNAd, is molecularly distinct from the 65-kD mucosal vascular addressin. The most abundant iodinated species by SDS-PAGE is 105 kD. When affinity isolated and immobilized on glass slides, MECA-79 immunoisolated material binds human and mouse lymphocytes avidly in a calcium dependent manner. Binding is blocked by mAb MECA-79, by antibodies against mouse or human LECAM-1 (the peripheral lymph node homing receptor, the MEL-14 antigen, LAM-1), and by treatment of PNAd with neuraminidase. Expression of LECAM-1 cDNA confers PNAd binding ability on a transfected B cell line. We conclude that LECAM-1 mediates lymphocyte binding to PNAd, an interaction that involves the lectin activity of LECAM-1 and carbohydrate determinants on the addressin.

The cDNA encoding baboon L-selectin was isolated from baboon peripheral blood lymphocytes-derived cDNA and its nucleotide (nt) sequence was determined. The deduced 372-amino acid (aa) sequence of baboon L-selectin is 95% identical to that of human L-selectin.

The neutrophil Mac-1 and gp100MEL-14 adhesion proteins are involved in neutrophil extravasation during inflammation. Both the expression and activity of Mac-1 are greatly increased after neutrophil activation. In contrast, neutrophils shed gp100MEL-14 from the cell surface within 4 minutes after activation with chemotactic factors or phorbol esters, releasing a 96-kilodalton fragment of the antigen into the supernatant. Immunohistology showed that gp100MEL-14 was downregulated on neutrophils that had extravasated into inflamed tissue. The gp100MEL-14 adhesion protein may participate in the binding of unactivated neutrophils to the endothelium; rapid shedding of gp100MEL-14 may prevent extravasation into and damage of normal tissues by activated neutrophils.

An endothelial cell surface molecule that is selectively expressed in mucosal organs is required for lymphocyte homing to mucosal lymphoid tissues. This 'vascular addressin' appears to function as a tissue-specific marker or address signal for recognition by lymphocytes circulating in the blood.

Leukocyte capture and rolling on the vascular endothelium is mediated principally by the selectin family of cell adhesion receptors. In a parallel plate flow chamber, neutrophil rolling on purified selectins or a selectin-ligand substrate was resolved by high speed videomicroscopy as a series of ratchet-like steps with a characteristic time constant (Kaplanski, G., C. Farnarier, O. Tissot, A. Pierres, A.-M. Benoliel, M. C. Alessi, S. Kaplanski, and P. Bongrand. 1993. Biophys. J. 64:1922-1933; Alon, R., D. A. Hammer, and T. A. Springer. 1995. Nature (Lond.). 374:539-542). Under shear, neutrophil arrests due to bond formation events were as brief as 4 ms. Pause time distributions for neutrophils tethering on P-, E-, L-selectin, or peripheral node addressin (PNAd) were compared at estimated single bond forces ranging from 37 to 250 pN. Distributions of selectin mediated pause times were fit to a first order exponential, resulting in a molecular dissociation constant (k(off)) for the respective selectin as a function of force. At estimated single bond forces of 125 pN and below, all three selectin dissociation constants fit the Bell and Hookean spring models of force-driven bond breakage equivalently. Unstressed k(off) values based on the Bell model were 2.4, 2.6, 2.8, 3.8 s(-1) for P-selectin, E-selectin, L-selectin, and PNAd, respectively. Bond separation distances (reactive compliance) were 0.39, 0.18, 1.11, 0.59 A for P-selectin, E-selectin, L-selectin, and PNAd, respectively. Dissociation constants for L-selectin and P-selectin at single bond forces above 125 pN were considerably lower than either Bell or Hookean spring model predictions, suggesting the existence of two regimes of reactive compliance. Additionally, interactions between L-selectin and its leukocyte ligand(s) were more labile in the presence of flow than the L-selectin endothelial ligand, PNAd, suggesting that L-selectin ligands may have different molecular and mechanical properties. Both types of L-selectin bonds had a higher reactive compliance than P-selectin or E-selectin bonds.

Tissue position-dependent or address-dependent expression of cell adhesion molecules has been proposed to play a part in cellular positioning in a variety of systems, for example during neural development, the metastasis of neoplasms, and the tissue-specific homing of lymphocytes. The extravasation of blood-borne lymphocytes is regulated by interactions with the endothelium of specialised venules, such as the high endothelial venules (HEY) in organized lymph nodes and mucosal lymphoid tissues1–4. At least three separate lymphocyte-HEV recognition systems have been described, one mediating tissue-selective lymphocyte interac-tions with HEV in peripheral lymph nodes, another in mucosal lymphoid organs, and a third in inflamed synovium4–8. We have previously identified a tissue-specific "vascular addressin’ in the mouse which is selectively expressed by venules mediating lym-phocyte-homing into mucosal tissues9. To determine whether this addressin is a specific adhesion molecule for lymphocytes, we have isolated it by monoclonal antibody-affinity chromatography and inserted it into supported phospholipid planar membranes10,11. Lymphocytes bind to membranes containing the addressin, but not to phospholipid bilayers or to control glycophorin-reconstituted membranes. Only those lymphocytes and lymphoma cell lines capable of binding to mucosal HEV adhere well to the isolated addressin; peripheral lymph node HEV-specific or HEV-non-binding cell lines do not bind. Binding is blocked by anti-addressin antibody MECA-367. We conclude that the mucosal vascular addressin is a tissue-specific endothelial cell-adhesion molecule for lymphocytes, and suggest that it could regulate lymphocyte traffic into mucosal tissues by mediating attachment of blood-borne cells to endothelium.