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Over the past two decades there have been tremendous advances in our understanding of tumor immunology, which have in turn led to new and exciting immunology-based therapeutics. However, further research is needed into the dynamics and regulation of the immune response in the tumor microenvironment in order to achieve the full potential of these agents in treating all cancer patients. Defining the role of cytokines, chemokines, and other soluble mediators will be essential to this endeavor. This chapter describes, in detail, the technical protocol and applicability of LEGENDplex™ bead-based multiplex assays in quantifying these critical signaling molecules.

Cytokine release syndrome (CRS) is a systemic inflammatory response syndrome caused by adverse effects of monoclonal or CAR T cell immunotherapy. CRS can also arise as a complication during infection in response to sepsis. Regardless of its initial etiology, CRS is characterized by an auto-amplifying cascade of leukocyte activation which drives the production and release of several inflammatory biomarkers. We have developed a murine-specific bead-based multiplex assay panel that simultaneously quantifies up to 13 CRS biomarkers from one sample volume aliquot using commonly available flow cytometers. This panel is manufactured with highly specific antibodies in order to ensure robust assay sensitivities, analytical accuracy, and reproducibility. The panel was validated with biological samples generated from both in-vitro and in-vivo models of inflammatory cytokine release. Trends in cytokine expression differences between treatment groups were orthogonally confirmed using single target ELISAs. Taken together, our cytokine release studies and rigorous performance validation establish this multiplex panel as a reliable tool for researchers seeking to mitigate the potentially fatal complications of CRS during infection and immunotherapy treatment.

Over the past two decades there have been tremendous advances in our understanding of tumor immunology, which have in turn led to new and exciting immunology-based therapeutics. However, further research is needed into the dynamics and regulation of the immune response in the tumor microenvironment in order to achieve the full potential of these agents in treating all cancer patients. Defining the role of cytokines, chemokines, and other soluble mediators will be essential to this endeavor. This chapter describes, in detail, the technical protocol and applicability of LEGENDplex™ bead-based multiplex assays in quantifying these critical signaling molecules.

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal allophycocyanin (APC) fluorescence intensity, are conjugated to antibodies specific to a particular analyte. Next, a selected panel of defined capture bead sets is incubated with a biological sample containing target analytes specific to the capture antibodies. A biotinylated detection antibody cocktail is added, which leads to the formation of capture bead-analyte-detection antibody sandwiches. Finally, streptavidin-phycoerythrin (SA-PE) is added, which binds to biotinylated detection antibodies, providing fluorescent signal intensities in proportion to the amount of bound analyte. The PE fluorescent signal of analyte-specific beads regions is quantified using flow cytometry, and the concentrations of particular analytes are determined using data analysis software and the standard curve generated in the assay. In this experiment, we use a mouse T helper cytokine panel to simultaneously quantify the concentration of 13 separate cytokine targets in tissue culture supernatants collected from mouse splenocytes cultured under various stimulatory conditions.

Immune checkpoint molecules are important regulators of immune activation. This regulation is critical to maintaining immune homeostasis, and is driven by a complex interplay of both co-stimulatory and inhibitory signals. These checkpoints are key to preventing autoimmunity in healthy individuals; however, cancer cells often exploit this system by modulating the expression of these molecules in order to suppress anti-tumor responses during host immuno-editing. The ability to accurately quantify expression levels of checkpoint molecules is critical to ongoing biomedical research efforts exploring potential therapeutic interventions to overcome tumor-mediated immune-evasion. To further aid these ongoing research efforts, we have developed a new bead-based multiplex assay panel specifically designed to quantify several human oncology-related immune checkpoint biomarkers including sCD25 (IL-2Ra), GITR, 4-1BB, sCD27, B7.2 (CD86), free active TGF-β1, CTLA-4, PD-L1, PD-L2, PD-1, TIM-3, LAG-3, Galectin-9. The panel utilizes fluorescence-encoded microsphere technology and is designed for use on commonly available flow cytometers. The panel has been extensively validated for specificity, dilution linearity, cross-reactivity, and inter- and intra-assay precision. This multiplex panel is a robust tool for measuring the concentrations of several immune checkpoint mediators in human serum, plasma, and cell culture supernatant samples, and offers greater efficiency and broader dynamic ranges compared to conventional ELISA-based quantification methods. Citation Format: Jason Lehmann, Priyanka Rughwani, Amy Zhao, Weiping Jiang, Shaoquan Ji, Binggang Sun. Novel bead-based multiplex assay for the quantification of human immune checkpoint biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4117.

Metastasis to distant organs is responsible for ~90% of cancer-related deaths. Adhesion molecules are cell surface proteins that mediate the interactions between disseminated tumor cells and the endothelium of blood vessels present in the tumor microenvironment, and thus play a major role in tumor metastasis. Serum and plasma samples from patients with metastatic disease (head, neck, breast, pancreatic, gastric, and lung cancer patients) or healthy controls were assayed with a bead-based multiplex immunoassay which measured the concentration levels of the following adhesion molecules: ICAM-1, ICAM-2, ICAM-3, VCAM-1, PECAM-1, ALCAM-1, EpCAM, NCAM, E-selectin, P-selectin, L-selectin, PSGL-1, and CD44. Although limited by sample size, differential concentrations of assay targets were detected between healthy and disease-state samples. We have demonstrated that this multiplex panel is a robust tool for measuring the concentration of a spectrum of adhesion molecules with great efficiency and convenience. This panel will help screen and identify the adhesion molecules that may be universally or specifically involved in metastasis of different types of cancer.

The inflammation of neuronal tissues is triggered by various stimuli such as traumatic brain injury, stroke, protein aggregation and pathogens. Neuroinflammation cues such as brain injury lead to protein release and microglia activation, which in turn activate glial cells. Activated glial cells produce both pro- and anti-inflammatory factors to regulate cell migration, growth, tissue repair or cell death. Neuroinflammation is involved in acute brain injury, stroke, and many neurodegenerative diseases, such as Alzheimers’s and Parkinson’s diseases, as well as disruption and permeability of the blood-brain barrier. Measurement of the cytokines, chemokines, growth factors and other neuronal disease biomarkers is essential in understanding the mechanisms of neuronal disease progression, but this is limited by the small sample size of CSF. It is therefore desirable to be able to measure a panel of important neuroinflammation biomarkers simultaneously. We have developed an assay panel targeting neuroinflammation biomarkers using fluorescence-encoded beads that are suitable for use on common lab flow cytometers. It allows for simultaneous quantification of the 13 key targets involved in neuroinflammation including VILIP-1, BDNF, free active TGF-β1, VEGF, IL-6, sTREM-1, β-NGF, IL-18, TNF-α, soluble RAGE, and CX3CL1 (Fractialkine). This panel has been developed with superior assay performance and specificity and has been validated by detecting expected values in biological samples including CSF samples from healthy and disease individuals. This high quality, low cost and easy to use panel will be a useful tool to the neuroinflammation research community.

Exposure to foreign material triggers host immune responses, which are primarily mediated by leukocytes including macrophages, neutrophils, dendritic cells, NK cells, T cells and B cells. These cells produce an array of soluble factors which coordinate host defense, tissue repair, and immunoregulatory functions. In addition, these cells and the factors they produce are also critically involved in various diseases including infection, autoimmune disorders, asthma, diabetes, and cancer. Therefore, quantification of these molecules in a cell type-specific context will help achieve in-depth understanding of the immune responses and disease processes. We have developed multiplex assay panels targeting specific mouse immune cells (CD4+ T helper cells, Macrophages/Microglia and B cells), using fluorescence–encoded beads that are suitable for use on common lab flow cytometers. Each panel allows for simultaneously quantification of 13 related analytes as shown in the table below. Cell TypePanel TargetsT helper cellsIL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 21, 22, IFN-γ, TNF-αMacrophages/MicrogliaIL-1β, 6, 10, 12p40, 12p70, 18, 23, TNF-α, CCL22/MDC, CCL17/TARC, CXCL1/KC, G-CSF, TGF-β1B cellsIL-2, 4, 6, 10, 12p70, 13, 17A, IFN-γ, TNF-α, BAFF, BCMA, sCD40 ligand, TGF-β1 Each antibody pair was carefully optimized for assay specificity, sensitivity, accuracy and reproducibility. In addition, these panels have been validated by detecting expected expression level changes of analytes in biological samples cultured under various stimulation conditions. These high quality, low cost and easy to use panels will be useful tools to the study of specific cells involved in innate and adaptive immune responses and the relevant diseases.

Immune responses are mediated by innate immune cells (e.g., macrophages) and subsequently, adaptive immune cells (e.g., T and B cells). Macrophages secrete an array of inflammatory mediators to affect their host defense, tissue repair, and immunoregulatory functions. Pro-inflammatory macrophages (M1) produce cytokines, such as IL-12p70, TNF-α, IL-6, IL-1β, IL-12p40, IL-18, IL-23, IFN-γ, and IP-10, while anti-inflammatory and tissue repairing macrophages (M2) release a different set of factors, such as IL-4, IL-10, IL-6, Arginase, TARC, MDC, IL-1RA and TGF-β1. We have developed new assay panels targeting human or mouse macrophage/microglia cells, using fluorescence–encoded beads that are suitable for use on commonly available flow cytometers. Each panel allows for the simultaneous quantification of 13 analytes related to macrophage function in humans and mice. These panels have been validated using biological samples to quantify analyte concentration changes in response to various stimuli. These high quality, low cost, easy-to-use panels provide an alternative multiplex solution to the biomedical research community.

In response to pathogens, especially viruses, cells release interferons and other cytokines to combat infection. Interferons are typically divided into three types: 1 (e.g., IFN-α, IFN-β), 2 (e.g., IFN-γ), and 3 (e.g., IFN-λ1, IFN-λ2). All inter-ferons are important for fighting viral infections and for regulating the immune system. In addition, interferons are critically involved in cancer and autoim-mune diseases such as psoriasis, systemic lupus erythematosus, and multiple sclerosis. Studying the expression profile of interferons and other related cyto-kines is essential to understanding the immune response against viral pathogens and other related disease processes. We have developed a multiplex panel, using fluorescence-encoded beads, which is suitable for use on commonly available flow cytometers. The panel simultaneously quantifies 13 mouse anti-viral immune proteins, including IFN-α, IFN-β, IFN-γ, CXCL1 (KC), TNF-α, CCL2 (MCP-1), IL-12p70, CCL5 (RANTES), IL-1β, CXCL10 (IP-10), GM-CSF, IL-10, and IL-6. The assay was validated for specificity, linearity, cross-reactivity, and inter and intra-assay precision. The assay was further validated using biological samples to quantify analyte concentration changes in response to poly (I:C) stimulation compared to untreated controls. This multiplex panel is a robust tool for measuring the concentration of anti-viral inflammatory mediators in murine biological samples, and offers greater efficiency and broader dynamic ranges compared to conventional ELISA assays.

There is substantial support for the hypothesis that T(H)1 cytokine responses are critical for the normal elaboration of allograft rejection. Recent studies by Wang et al. (1) underscore the importance of T(H)2 responses in xenograft rejection and revealed that T(H)1 cytokines, IL-12 and interferon-gamma (IFN-gamma), can negatively regulate the development of humoral responses necessary for xenograft rejection. Their exceptional studies prompted us to test whether the ability of allografts to elicit cellular rejection and xenografts to induce humoral rejection also result from the differential ability to induce T(H)1 and T(H)2 responses. We compared the kinetics of antibody and cytokine (IFN-gamma and IL-4) production in C57BL/6 mice following allograft transplantation with BALB/c hearts and in C57BL/6 and BALB/c mice following transplantation with Lewis rat hearts. We also compared the ability of BALB/c mice, deficient in the ability to produce IL-4 or IFN-gamma, to reject xenografts and produce xenoantibodies. We observed that T(H)1/T(H)2 cytokine production minimally affected the kinetics of graft rejection but regulated the magnitude of IgG subclass production. Anti-graft IgM played a critical role in initiating acute antibody-mediated xenograft rejection, and the production antigraft IgM was unaffected by IL-4 or IFN-gamma deficiency. In contrast to the report by Wang et al. (1), we conclude that antibody-mediated xenograft rejection in the concordant Lewis rat heart-to-C57BL/6 mouse xenotransplantation model is dependent on anti-IgM production but independent of T(H) cytokine profiles.

Immune responses are mediated by innate immune cells (e.g., macrophages) and subsequently, adaptive immune cells (e.g., B cells). Macrophages secrete an array of cytokines to effect their functions in host defense, tissue repair and immunoregulation. Pro-inflammatory macrophages (M1) produce cytokines, such as IL-12p70, TNF-α, IL-6, IL-1β, IL-12p40, IL-23, IFN-γ, and IP-10, while anti-inflammatory and tissue repairing macrophages (M2) release a different set of factors, such as IL-4, IL-10, IL-6, Arginase, TARC and IL-1RA. B cells, in addition to antibody production, also secrete an array of cytokines that mediate Th1- and Th2-like immune responses. These cytokines are produced by regulatory B cells (e.g., IL-10, TGF-β1) and B effector cells, namely Be1 (e.g., TNF-α, TNF-β, IFN-γ, and IL-12p70) and Be2 cells (e.g., IL-2, IL-4, IL-6, TNF-α, and IL-13). Other cytokines associated with activation and survival of B cells such as APRIL, BAFF, and CD40L are also important targets in B cell related processes. We have developed new assay panels targeting macrophage/microglia and B cells, using fluorescence–encoded beads that are suitable for use on general lab flow cytometers. Each panel allows simultaneous quantification of 13 related analytes as shown below. PanelTargetMacrophage/MicrogliaIL-1β, 1RA, 4, 6, 10, 12p40, 12p70, 23, IFN-γ, TARC, TNF-α, Arginase, IP-10B cellIL-2, 4, 6, 10, 12p70, 13, 17A, IFN-γ, TNF-α, TNF-β, BAFF, APRIL, CD40L These panels have been validated by detecting expected changes in biological samples. These high quality, low cost and ease of use panels provide an alternative multiplex solution to the biomedical research community.

The relationship between obese gene expression and energy intake was determined in pigs of various body weights. With ad libitum consumption, expression increased (P0.001) with body weight from 55 to 163 kg. Obese mRNA relative abundance was correlated with fat mass (r = 0.74, P0.0001) and percentage of fat (r = 0.72, P0. 0001). Obese expression was also evaluated at 159 kg (initial weight) and ad libitum, maintenance or 23% of maintenance intake for 28 d. Obese mRNA was independent of treatment (P0.78) despite considerable weight differences. Obese mRNA abundance was then compared at 136 kg (initial weight) and ad libitum or maintenance intake for 3 or 28 d. Abundance was not influenced by either duration of treatment or intake, despite a small increase (P0.01) in serum nonesterified fatty acids (NEFA) and a reduction (P0.02) in insulin attributable to maintenance intake. Finally, mRNA abundance was determined at 60 and 136 kg and conditions of food deprivation or ad libitum intake for 3 d. Food deprivation reduced (P0.01) serum insulin and increased (4- to 5-fold) NEFA concentrations. Obese mRNA abundance was greater (P0.01) in the heavier pigs and was reduced (P0.01) by food deprivation. We conclude that obese mRNA abundance in pigs correlates with fat mass and percentage of body fat under conditions of ad libitum intake. Furthermore, obese mRNA abundance is reduced by food deprivation, whereas lesser degrees of intake restriction do not change obese mRNA abundance, even when accompanied by appreciable weight loss.

The product of the obese gene, leptin, may be an important regulator of adiposity via its regulation of feed intake and energy metabolism. Probes were developed using the polymerase chain reaction to analyze gene expression and determine the structure of the porcine ob gene. Porcine ob was expressed in adipose tissue as a 3,100 bp mRNA. Finished pigs (136 kg) had higher (P

We investigated associations between type 2 diabetes (DM) and several variables, including poor oral health and overweight/obesity, among a group of elderly Hmong subjects (60 years and older) who emigrated to the United States following the Vietnam conflict. Each subject was interviewed and their weight, height and waist circumference were measured. Each subject had an oral health examination. Each subject's saliva was analyzed for seven components related to inflammation. The presence of DM was correlated with poor oral health (POH) and overweight/obesity (OW) separately. There was a strong correlation between concurrent POH and OW and the presence of DM: all subjects with both POH and OW had DM. Logistic multivariate analysis of OW, POH, age, years of residence in California, and stress level revealed a significant association between the presence of DM and concurrent OW and POH. A change in diet after immigration was excluded as an explanatory variable. Subjects with DM and concurrent OW and POH had significantly elevated salivary levels of five analyses related to chronic inflammation. The association between POH and OW and the presence of DM needs further study.

Seven-day-old primary myotube cultures derived from embryonic chicken limb muscles were used to determine the effects of the beta-adrenergic agonist isoproterenol (ISO) on muscle protein metabolism in vitro. Isoproterenol increased (P less than .05) total protein accumulation after 2 h of acute exposure and after chronic exposure for 24 and 48 h. Isoproterenol did not consistently retard rate of protein degradation of the total protein (TP), myofibrillar protein (MFP) pools, and myosin heavy-chain subunit (MHC); degradation of these protein pools tended to be slowed by inclusion of ISO in the culture medium. After acute treatment of 1 X 10(-4) M ISO for 2 h, TP, but not MFP and MHC, synthesis rate was increased, and after chronic exposure to 1 X 10(-4), 1 X 10(-5), and 1 X 10(-6) M ISO, TP, MFP, and MHC synthesis rates and net accumulation of TP, cytoplasmic protein, and MHC fractions were enhanced (P less than .05). The beta-adrenergic antagonist propranolol (1 X 10(-5) M) blocked chronic stimulatory effects of ISO. Furthermore, after 48 h of exposure to ISO, effects on protein synthesis were less pronounced than those observed after 24 h of exposure. Isoproterenol imparted a more pronounced effect on protein synthesis than on protein degradation, indicating that increased muscle protein accretion observed in animals after ISO treatment is likely a function of enhanced protein synthesis.

Macrophages can be both beneficial and detrimental after CNS injury. We previously showed rapid accumulation of macrophages in injured immature brain acutely after ischemia-reperfusion. To determine whether these macrophages are microglia or invading monocytes, we subjected post-natal day 7 (P7) rats to transient 3 h middle cerebral artery (MCA) occlusion and used flow cytometry at 24 and 48 h post-reperfusion to distinguish invading monocytes (CD45high/CD11b+) from microglia (CD45low/medium/CD11b+). Inflammatory cytokines and chemokines were determined in plasma, injured and contralateral tissue 1-24 h post-reperfusion using ELISA-based cytokine multiplex assays. At 24 h, the number of CD45+/CD11b+ cells increased 3-fold in injured compared to uninjured brain tissue and CD45 expression shifted from low to medium with less than 10% of the population expressing CD45high. MCA occlusion induced rapid and transient asynchronous increases in the pro-inflammatory cytokine IL-beta and chemokines cytokine-induced neutrophil chemoattractant protein 1 (CINC-1) and monocyte-chemoattractant protein 1 (MCP-1), first in systemic circulation and then in injured brain. Double immunofluorescence with cell-type specific markers showed that multiple cell types in the injured brain produce MCP-1. Our findings show that despite profound increases in MCP-1 in injured regions, monocyte infiltration is low and the majority of macrophages in acutely injured regions are microglia.

Signaling through the PGI2 receptor (IP) has been shown to inhibit inflammatory responses in mouse models of respiratory syncytial viral infection and OVA-induced allergic responses. However, little is known about the cell types that mediate the anti-inflammatory function of PGI2. In this study, we determined that PGI2 analogs modulate dendritic cell (DC) cytokine production, maturation, and function. We report that PGI2 analogs (iloprost, cicaprost, treprostinil) differentially modulate the response of murine bone marrow-derived DC (BMDC) to LPS in an IP-dependent manner. The PGI2 analogs decreased BMDC production of proinflammatory cytokines (IL-12, TNF-α, IL-1α, IL-6) and chemokines (MIP-1α, MCP-1) and increased the production of the anti-inflammatory cytokine IL-10 by BMDCs. The modulatory effect was associated with IP-dependent up-regulation of intracellular cAMP and down-regulation of NF-κB activity. Iloprost and cicaprost also suppressed LPS-induced expression of CD86, CD40, and MHC class II molecules by BMDCs and inhibited the ability of BMDCs to stimulate Ag-specific CD4 T cell proliferation and production of IL-5 and IL-13. These findings suggest that PGI2 signaling through the IP may exert anti-inflammatory effects by acting on DC.

Due to the immunological and physiological similarities between non-human primates (NHPs) and humans, understanding the disease progression and immune response in NHP species can predict response in humans. Therefore, NHPs are preferred model systems in drug safety test, vaccine development, and infectious disease and brain research. Correspondingly, accurate measurement of soluble biomarkers in NHPs is important for drug development and biomedical research. We have developed several NHP multi-analyte flow assay panels, including T Helper Cytokine Panel, Inflammation Panel, Growth Factor Panel, Adipokine Panel, and Cytokine/Chemokine Panel, for the quantification of a variety of NHP biomarkers including cytokines (IL-1 β , IL-2, 4, 5, 6, 10, IL-12p40, 17A, IL-17F, IL-21, IL-22, IL-23, IL-33, IFN- γ , TNF- α ) , chemokines (RANTES, MCP-1, MIP-1 α , MIP-1 β , IP-10, IL-8, MIG and I-TAC), growth factors (EGF, Ang-2, G-CSF, GM-CSF, PDGF-AA, PDGF-BB, FGF-b, etc.) and adipokines (Adiponectin, Adiosin, Resistin, Leptin, etc.). These assays are bead-based sandwich immunoassays, with fluorescence-encoded beads that can be separated by a variety of flow cytometers. In each panel, up to thirteen NHP biomarkers can be quantified simultaneously. Each panel has been validated for use in cell culture supernatant, serum, plasma and urine samples for both rhesus and cynomolgus monkey species. These panels provide higher sensitivities and broader dynamic ranges than traditional ELISA methods, offering useful tools for researchers dedicated to NHP research and drug discovery.

In response to pathogens, especially viruses, cells release interferons and other cytokines to fight the infections. Interferons are typically divided into three types: I (e.g., INF-α, IFN-β), II (e.g., IFN-γ), and III (e.g., IFN-λ1, IFN-λ2). All interferons are important for fighting viral infections and for regulating the immune system. In addition, interferons are critically involved in cancer and autoimmune diseases such as psoriasis, systemic lupus erythematosus, and multiple sclerosis. Expression profiling of interferons and other related cytokines is critical in achieving a deeper understanding of the immune responses and various disease processes. We have developed a multiplex Human Interferon Panel, using fluorescence-encoded beads that are suitable for use on various flow cytometers. This panel allows simultaneous quantification of 13 cytokines involved in anti-virus responses including interferons (α, β, γ, λ1 and λ2), interleukins (1β, 6, 8, 10, 12), TNF-α, IP-10 and GM-CSF. Each antibody pair was carefully selected for assay specificity, sensitivity, accuracy and reproducibility. The panel has been validated by detecting expected changes in biological samples. Further advantages include high sensitivity, small sample volume, flexible assay configurations, and time- and cost-effectiveness. The Interferon Panel can be used for serum, plasma, cell culture supernatant and other sample types, offering a useful tool for biomedical research and drug discovery.

Nonselective cyclooxygenase (COX) inhibition during the development of allergic disease in a murine model causes an increase in type 2 cytokines and lung eosinophilia; however, the mechanisms responsible for this augmented allergen-induced inflammation have not been examined. Ab depletion of CD4 and CD8 cells revealed that the heightened allergic inflammation caused by COX inhibition was CD4, but not CD8, dependent. Allergen sensitization and airway challenge alone led to undetectable levels of IL-5 and IL-13 in the lungs of IL-4, IL-4Rα, and STAT6 knockout (KO) mice, but COX inhibition during the development of allergic inflammation resulted in wild-type levels of IL-5 and IL-13 and heightened airway eosinophilia in each of the three KO mice. These results indicate that the effect of COX inhibition was independent of signaling through IL-4, IL-4Rα, and STAT6. However, whereas COX inhibition increased IgE levels in allergic wild-type mice, IgE levels were undetectable in IL-4, IL-4Rα, and STAT6 KO mice, suggesting that IL-13 alone is not a switch factor for IgE synthesis in this model. These results illustrate the central role played by products derived from the COX pathway in the regulation of allergic immune responses.

Current multiplex assay technologies on the market for soluble targets are frequently associated with high reagent and instrument cost, inconsistent assay performance, inaccurate data analysis, and lack of good reproducibility. To address the issues, we developed four multiplexed bead-based assay panels for simultaneous quantification of soluble markers using standard flow cytometers available in many laboratories. Our human and mouse T help (Th) panels allow quantification of 13 cytokines, including interleukins (IL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 21, 22), IFN-g and TNF- α , which are collectively secreted by Th1, Th2, Th9, Th17, Th22 and T follicular cells. Our human and mouse proinflammatory chemokine panels detect 13 chemokines, including MCP-1 (CCL2), RANTES (CCL5), IP-10 (CXCL10), Eotaxin (CCL11), TARC (CCL17), MIP-1 α (CCL3), MIP-1 β (CCL4), MIG (CXCL9), and MIP-3 α (CCL20). The human chemokine panel also includes ENA-78 (CXCL5), GRO α (CXCL1), I-TAC (CXCL11), and IL-8 (CXCL8), and the mouse chemokine panel contains LIX (CXCL5), KC (CXCL1), BLC (CXCL13) and MDC (CCL22). Each antibody pair was carefully selected for assay specificity, sensitivity, accuracy and reproducibility. The assay panels have been validated in appropriate biological samples, detecting changes as expected. Compared with similar assay products on the market, LEGENDplex™ assays provide equal or better analytical performance with reduced cost, better performance consistency, and greater flexibility. To facilitate analysis of data (FCS 2.0, 3.0, 3.1, and list mode files) from different flow cytometers, a software package with an intuitive interface, accurate curve-fitting, and robust reporting was developed and is offered free of charge. The assays and software can be used for quantitative and qualitative analysis of soluble biomarkers from serum, plasma, cell culture supernatant, and other sample types, offering a more economical alternative for multiplex solutions.

T helper or “Th” cells, play important roles in regulating the responses of the immune system. They secrete cytokines to stimulate various effector cells, such as cytotoxic T cells, B cells and macrophages. Accurate measurement of Th cytokine expression is critical to identify the corresponding Th cell responses and to develop in depth understanding of the immune responses. We have developed two multiplexed assay panels, i.e., the human and mouse Th panels, using fluorescence-encoded beads that are suitable for use on various flow cytometers. Both panels allow simultaneous quantification of 13 human and mouse cytokines including interleukins (IL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 21, 22), IFN-g and TNF-α, which are collectively secreted by Th1, Th2, Th9, Th17, Th22 or T follicular cells. Each antibody pair was carefully selected for assay specificity, sensitivity, accuracy and reproducibility. The assays provide higher detection sensitivity and broader dynamic ranges than traditional ELISA methods. The panels have been validated by detecting expected changes in biological samples. Further advantages include small sample volume, flexible assay configurations, and time- and cost-effectiveness. The Th cytokine panels can be used for serum, plasma, cell culture supernatant and other sample types, offering useful tools for biomedical research and drug discovery.

The isoflavones, genistein and genistin, are cytotoxic in vitro (e.g. , inhibition of cell proliferation), due in part to inhibition of protein tyrosine kinase and DNA topoisomerase activities. Normal cell functions associated with these enzymatic activities could potentially be impaired in animals through ingestion of soybean products. In this study, cultured rat myogenic cells (L8) were used to determine whether genistein or genistin influences myoblast proliferation and fusion, and myotube protein synthesis and degradation. Genistein or genistin was dissolved in dimethylsulfoxide and included in the culture medium at 0, 1, 10 or 100 micromol/L. Myoblast proliferation was measured by methyl-3H-thymidine incorporation over 48 h. Myoblast differentiation was evaluated by the number of nuclei in multinucleated myotubes. Myotube protein synthesis was measured by 2-h 3H-amino acid incorporation into the myosin and total protein pools after acute (2 h) or chronic (24 h) exposure to similar treatments; protein degradation was measured by measuring radioactivity in protein pools following a time course of protein breakdown after myotube proteins were prelabeled with 3H-amino acids. Genistein or genistin strongly inhibited in vitro myoblast proliferation (P < 0.001) and fusion (P < 0.001) in a dose-dependent manner with effective genistein concentration as low as 1 micromol/L. Genistein or genistin inhibited protein accretion in myotubes (P < 0.001). Decreased protein accretion is largely a result of inhibition on cellular (myofibrillar) protein synthesis rate. No adverse effect on protein degradation was observed. Results suggest that if sufficient circulating concentrations are reached in tissues of animals consuming soy products, genistein/genistin can potentially affect normal muscle growth and development.

The physiologic response to infection includes reductions in tissue concentrations of anabolic growth factors as a means of reducing growth and conserving nutrients for immunologic processes. This repartitioning of nutrients is accompanied by anorexia, which has been linked to increased leptin expression. Furthermore, leptin and growth hormone (GH) concentrations are inversely related, with leptin being required for normal GH release. The objective of this study was to determine if pretreatment with GH would influence endotoxin-induced changes in leptin expression or attenuate endotoxin-induced reductions in serum insulin-like growth factor-1 (IGF-1) and IGF-1 expression in liver and longissimus muscle. In experiment 1, 40 pigs were assigned to four treatments (n = 10 per treatment) arranged as a 2x2 factorial with GH (s.c. injection, 2 mg 1 h before challenge and 2 mg 2 h after challenge) and endotoxin (single i.m. injection, 25 microg/kg body weight) as main effect variables. Pretreatment with GH resulted in a marked increase (p0.001) in serum GH within 1 h that was sustained throughout the study. Endotoxin challenge reduced (p0.003) serum IGF-1 independent of GH (GH x endotoxin, p0.682), and reduced (p0.05) IGF-1 expression in longissimus muscle but not liver. Leptin mRNA abundance was reduced 56% (p0.005) by GH but was not affected by endotoxin (p0.81). In experiment 2, 36 pigs (n = 12 per treatment) were either allowed ad libitum feed consumption with no injection or deprived of feed and injected twice with either saline or endotoxin 24 h apart. Feed deprivation reduced leptin expression (p0.05). However, endotoxin did not change leptin expression but markedly increased (p0.05) serum haptoglobin. These data indicate that changes in IGF-1 status in endotoxin-challenged pigs are independent of serum GH and that leptin expression is not increased by endotoxin challenge in the pig. These data also indicate a regulatory linkage between GH and leptin in vivo.

The objective of this study was to establish the developmental pattern and tissue specificity of porcine myostatin expression and to evaluate expression in skeletal muscle during circumstances in which muscle growth was altered. Northern blot analysis revealed two transcripts (1.5 and 0.8 kb). Myostatin mRNA was detected in whole fetuses at 21 and 35 days and was markedly increased ( P < 0.05) by 49 days. At birth, mRNA abundance in longissimus muscle had declined significantly ( P < 0.05) from that at day 105 of gestation and continued to decrease ( P < 0.05) to its lowest level 2 wk postnatally (4 kg body wt). Myostatin expression was higher ( P < 0.05) at 55, 107, and 162 kg body wt than at 4 kg body wt. Postnatally, myostatin mRNA was detected in skeletal muscle and mammary gland. Expression at birth was 65% higher ( P < 0.04) in longissimus muscle of low-birth-weight piglets (0.57 ± 0.052 kg body wt) vs. normal (1.37 ± 0.077 kg body wt) littermates, irrespective of gender. However, suppression of longissimus muscle growth by food deprivation (3 days) did not alter ( P > 0.15) myostatin expression in either 4- or 7-wk-old piglets. Additionally, myostatin mRNA abundance was not changed by porcine growth hormone administration in growing animals. These data indicate that myostatin expression in skeletal muscle peaks prenatally and that greater expression is associated with low birth weight. Expression in mammary gland indicates a possible role for myostatin in mammary gland development and/or lactation.

The product of the leptin (i.e., obese) gene may be an important regulator of energy metabolism, adiposity, and reproduction, and is perhaps linked to meat quality determinants such as marbling. Molecular probes were developed using polymerase chain reaction (PCR) technology to evaluate leptin expression in adipose depots and to evaluate the tissue-dependent nature of expression reported in other species. A 438 bp fragment representing the coding region of the bovine leptin gene excluding the N-terminal secretory signal was amplified, cloned into a plasmid vector (pASK75), and expressed in E. coli. Sequence analysis of the cDNA and the corresponding polypeptide indicate that, overall, both share approximately 87% homology with the mouse and human leptin genes and polypeptides. Amino terminal sequencing (30 amino acid residues) of the recombinant bovine leptin (rBL) protein revealed 100% homology with mouse and human leptin. The bovine leptin gene is expressed as a 3,090 nt mRNA which is detected in adipose tissue, but is not found in brain (despite the appreciable fat content and lipid metabolism) or other tissues. Leptin gene expression in several adipose depots (subcutaneous, renal, and omental) was similar (P = .73) in finished cattle.

The description of a new subpopulation of IL-17 producing T helper cells opened a new avenue in the understanding of immune mechanisms and immune pathologies. This recently described subpopulation has been called Th17 cells, and has a different cytokine profile from the previously described subpopulations Th1 and Th2. Th1 produce IFNγ and Th2 produce IL-4, IL-5, IL-6, and IL-13. Murine Th17 cells produce a large series of cytokines: IL-17A, IL-17F, IL-21, IL-22, IL-26, IL-6 and tumor necrosis factor (TNF)α. In humans, IL-17A, IL-17F, IL-21, IL-22 have been identified as effector cytokines that are preferentially produced by Th17 cells. In this study, we describe bioassays for cytokines produced by Th17 cells. IL-17A activity was measured by its ability to induce IL-6 in human dermal fibroblast (HDF). HDF were incubated with serial dilutions of IL-17A for 48 h, and the IL-6 was measure by ELISA. hIL-17A has ED50 of 2-4 ng/ml. IL-22 bioactivity was measure using human Colo-205 cells. IL-22 is known to induce IL-10 production in endothelial cells. Colo-205 cells were treated with IL-22 for 48 h, and IL-10 was measured by ELISA. hIL-22 shows an ED50 of 0.062-0.177 ng/ml. Mouse IL-21 was measured in a proliferation assay using CTLL-2. The ED50 for mouse IL-21 is 40 ng/ml. We have successfully expressed these cytokines and shown that they have potent biological activity. BioLegend.

Human thymic stromal lymphopoietin (TSLP) is a 24 kDa monomeric glycoprotein produced primarily by non-hematopoietic cells such as fibroblasts, epithelium, and stromal cells. TSLP induces myeloid cells to release T cell chemoattactents while enhancing maturation of CD11c+ dendritic cells that in turn promote a Th2 immune phenotype. We have developed a sandwich ELISA to quantitatively measure human TSLP concentrations in culture media and human serum. The capture and detection antibodies consist of antigen affinity-purified goat anti-human TSLP polyclonal antibodies generated from recombinant human TSLP as the immunogen. The assay has a linear detection range from 31 to 250 pg/ml, and a threshold of detection at approximately 2 pg/ml. Serum spike recovery averaged 92.6% for four human serums (2 male, 2 female) for spike concentrations from 31.25pg to 500pg/ml inclusive. Assay robustness was further demonstrated by acceptable linearity of sample dilutions, intra-assay and inter-assay precisions. When primary normal human bronchial epithelial cells were stimulated with dsRNA and/or IL-4, TSLP concentration in the culture supernatant of the double stimulated cells increased significantly (53pg/ml, sd 3.37) compared with that from dsRNA stimulated cells (22pg/ml, sd 1.5, p=0.001). Unstimulated cell media had no detectable TSLP. This assay will provide a valuable resource for the study of role of TSLP in normal and pathophysiological conditions.

The incidence of neonatal stroke is high and currently there are no strategies to protect the neonatal brain from stroke or reduce the sequelae. Agents capable of modifying inflammatory processes hold promise. We set out to determine whether delayed administration of one such agent, minocycline, protects the immature brain in a model of transient middle cerebral artery (MCA) occlusion in 7-day-old rat pups. Injury volume in minocycline (45 mg/kg/dose, beginning at 2 h after MCA occlusion) and vehicle-treated pups was determined 24 h and 7 days after onset of reperfusion. Accumulation of activated microglia/macrophages, phosphorylation of mitogen-activated protein kinase (MAPK) p38 in the brain, and concentrations of inflammatory mediators in plasma and brain were determined at 24 h. Minocycline significantly reduced the volume of injury at 24 h but not 7 days after transient MCA occlusion. The beneficial effect of minocycline acutely after reperfusion was not associated with changed ED1 phenotype, nor was the pattern of MAPK p38 phosphorylation altered. Minocycline reduced accumulation of IL-1β and CINC-1 in the systemic circulation but failed to affect the increased levels of IL-1β, IL-18, MCP-1 or CINC-1 in the injured brain tissue. Therefore, minocycline provides early but transient protection, which is largely independent of microglial activation or activation of the MAPK p38 pathway.

Cyclooxygenase (COX) inhibition during allergic sensitization and allergen airway challenge results in augmented allergic inflammation. We hypothesized that this increase in allergic inflammation was dependent on increased generation of leukotrienes that results from COX inhibition, as leukotrienes are important proinflammatory mediators of allergic disease. To test this hypothesis, we allergically sensitized and challenged mice deficient in 5-lipoxygenase (5-LO). We found that 5-LO knockout mice that were treated with a COX inhibitor during allergic sensitization and challenge had significantly increased airway hyperresponsiveness (AHR) (p < 0.01) and airway eosinophilia (p < 0.01) compared with 5-LO knockout mice that were treated with vehicle. The proinflammatory cytokines have also been hypothesized to be critical regulators of airway inflammation and AHR. We found that the increase in airway eosinophilia seen with COX inhibition is dependent on IL-5, whereas the increase in AHR is not dependent on this cytokine. In contrast, the COX inhibition-mediated increase in AHR is dependent on IL-13, but airway eosinophilia is not. These results elucidate the pathways by which COX inhibition exerts a critical effect of the pulmonary allergen-induced inflammatory response and confirm that COX products are important regulators of allergic inflammation.

The objective of this study was to determine whether the improvements in growth and efficiency of gain achieved by recombinant porcine somatotropin (pST) are associated with altered expression of the p94, calpastatin, or alpha-actin genes in porcine longissimus (LD) muscle. Forty-eight barrows (initial 64.2 to 67.4 kg BW) were assigned to four treatments (n = 12) arranged as a 2 x 2 factorial in a randomized complete block design. Factors were duration of treatment (3 or 6 wk) and pST administration (0 or 3 mg x pig(-1) x d(-1)). Plasma samples were obtained 24 h after the first pST injection and at the end of the each treatment period for assays of selected variables. The LD samples were obtained at 3 and 6 wk of pST treatment. Northern blot analysis of calpastatin expression in LD muscle revealed three distinct transcription products of approximately 8.5 (CPST I), 5.5 (CPST II), and 2.5 (CPST III) kb; CPST II was reduced (P.02) 33 and 61% by pST at 3 and 6 wk, respectively, whereas CPST I and III were not influenced (P.12). Neither alpha-actin nor p94 was responsive to pST injection. As expected, pST resulted in higher (50%, P.02) plasma insulin within 24 h and one- and twofold higher (P.01) concentrations at 3 and 6 wk, respectively. Glucose was increased (P.01) at 3 (15%) and 6 (10%) wk, whereas urea nitrogen was reduced (32 to 36%, P.01). The efficacy of pST was evident in that ADG was improved (P.01) 11 to 13% independent of time. Likewise, feed intake was reduced (P.01) 10 to 11% and gain: feed improved (P.01) approximately 26% for pigs receiving pST independent of time. These data indicate that the enhanced muscle growth achieved by pST is not associated with altered expression of p94 or alpha-actin, or an increase in the abundance of any calpastatin transcription product.

The ID family of helix-loop-helix proteins regulates cell proliferation and differentiation in many different developmental pathways, but the functions of ID4 in mammary development are unknown. We report that mouse Id4 is expressed in cap cells, basal cells and in a subset of luminal epithelial cells, and that its targeted deletion impairs ductal expansion and branching morphogenesis as well as cell proliferation induced by estrogen and/or progesterone. We discover that p38MAPK is activated in Id4-null mammary cells. p38MAPK is also activated following siRNA-mediated Id4 knockdown in transformed mammary cells. This p38MAPK activation is required for the reduced proliferation and increased apoptosis in Id4-ablated mammary glands. Therefore, ID4 promotes mammary gland development by suppressing p38MAPK activity.