LEGENDplex™: Bead-assisted multiplex cytokine profiling by flow cytometry.

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.

Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform.

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.

Synergism between obesity and poor oral health associated with diabetes in an elderly human population.

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.

Leukotriene A(4) hydrolase inhibition attenuates allergic airway inflammation and hyperresponsiveness.

RATIONALE: Allergic asthma is characterized by reversible airway obstruction, lung inflammation, and airway hyperresponsiveness (AHR). Previous studies using leukotriene B(4) (LTB(4)) receptor 1-deficient mice and adoptive transfer experiments have suggested that LTB(4) plays a role in lung inflammation and AHR. OBJECTIVES: In this study, we used a leukotriene A(4) hydrolase (LTA(4)H) inhibitor as a pharmacological tool to directly examine the role of LTB(4) in a mast cell-dependent murine model of allergic airway inflammation. METHODS: We used the forced oscillation technique to test the effects of an LTA(4)H inhibitor dosed during the challenge phase on AHR. Lung tissue and lavage were collected for analysis. MEASUREMENTS AND MAIN RESULTS: Treatment with an LTA(4)H inhibitor improved multiple parameters encompassing AHR and lung function. Significant decreases in inflammatory leukocytes, cytokines, and mucin were observed in the lung lumen. Serum levels of antigen-specific IgE and IgG1 were also decreased. Labeled antigen uptake by lung dendritic cells and subsequent trafficking to draining lymph nodes and the lung were decreased on LTA(4)H inhibitor treatment. Provocatively, inhibition of LTA(4)H increased lipoxin A(4) levels in lung lavage fluid. CONCLUSIONS: These data suggest that LTB(4) plays a key role in driving lung inflammation and AHR. Mechanistically, we provide evidence that inhibition of LTA(4)H, affects recruitment of both CD4(+) and CD8(+) T cells, as well as trafficking of dendritic cells to draining lymph nodes, and may beneficially modulate other pro- and antiinflammatory eicosanoids in the lung. Inhibition of LTA(4)H is thus a potential therapeutic strategy that could modulate key aspects of asthma.

Regulation of contractile vacuole formation and activity in Dictyostelium.

The contractile vacuole (CV) system is the osmoregulatory organelle required for survival for many free-living cells under hypotonic conditions. We identified a new CV regulator, Disgorgin, a TBC-domain-containing protein, which translocates to the CV membrane at the late stage of CV charging and regulates CV-plasma membrane fusion and discharging. disgorgin(-) cells produce large CVs due to impaired CV-plasma membrane fusion. Disgorgin is a specific GAP for Rab8A-GTP, which also localizes to the CV and whose hydrolysis is required for discharging. We demonstrate that Drainin, a previously identified TBC-domain-containing protein, lies upstream from Disgorgin in this pathway. Unlike Disgorgin, Drainin lacks GAP activity but functions as a Rab11A effector. The BEACH family proteins LvsA and LvsD were identified in a suppressor/enhancer screen of the disgorgin(-) large CV phenotype and demonstrated to have distinct functions in regulating CV formation. Our studies help define the pathways controlling CV function.

Leukotriene B4 receptors BLT1 and BLT2: expression and function in human and murine mast cells.

Leukotriene B4 (LTB4) is a potent activator and chemoattractant for leukocytes and is implicated in several inflammatory diseases. The actions of LTB4 are mediated by two cell surface receptors, BLT1, which is predominantly expressed in peripheral blood leukocytes, and BLT2, which is expressed more ubiquitously. Recently, BLT1 expression and LTB4-dependent chemotaxis have been reported in immature mast cells (MCs). We now show the first evidence for BLT2 mRNA expression, in addition to BLT1, in murine bone marrow-derived MCs (mBMMCs) and in a human MC line (HMC-1). Protein expression of BLT1 was confirmed by mAb staining in HMC-1 cells and shown to be predominantly intracellular. Both HMC-1 cells and mBMMCs migrated to LTB4 in a dose-dependent manner in chemotaxis assays. Migration to LTB4 could be inhibited by either a BLT1- or BLT2-selective antagonist. Significant dose-dependent migration of mBMMCs also was observed to 12-(S)-hydroxyeicosotetraenoic acid, a BLT2-selective agonist, demonstrating functional BLT2 activity in these cells. Stimulation of mBMMCs with LTB4 induced transient, dose-dependent, ERK phosphorylation and changes in Akt phosphorylation. Dose-dependent ERK phosphorylation also was observed in response to 12-(S)-hydroxyeicosotetraenoic acid, indicating signaling downstream of BLT2. Pretreatment of mBMMCs with stem cell factor significantly down-regulated expression of BLT1 and BLT2 mRNA and inhibited their migration to LTB4. This study demonstrates expression of functional LTB4 receptors, both BLT1 and BLT2, in murine and human MCs and a regulatory role for stem cell factor in their expression. These receptors may mediate recruitment and accumulation of MCs in response to LTB4 production in areas of inflammation.

SPAK kinase is a substrate and target of PKCtheta in T-cell receptor-induced AP-1 activation pathway.

Protein kinase C-theta (PKCtheta) plays an important role in T-cell activation via stimulation of AP-1 and NF-kappaB. Here we report the isolation of SPAK, a Ste20-related upstream mitogen-activated protein kinase (MAPK), as a PKCtheta-interacting kinase. SPAK interacted with PKCtheta (but not with PKCalpha) via its 99 COOH-terminal residues. TCR/CD28 costimulation enhanced this association and stimulated the catalytic activity of SPAK. Recombinant SPAK was phosphorylated on Ser-311 in its kinase domain by PKCtheta, but not by PKCalpha. The magnitude and duration of TCR/CD28-induced endogenous SPAK activation were markedly impaired in PKCtheta-deficient T cells. Transfected SPAK synergized with constitutively active PKCtheta to activate AP-1, but not NF-kappaB. This synergistic activity, as well as the receptor-induced SPAK activation, required the PKCtheta-interacting region of SPAK, and Ser-311 mutation greatly reduced these activities of SPAK. Conversely, a SPAK-specific RNAi or a dominant-negative SPAK mutant inhibited PKCtheta- and TCR/CD28-induced AP-1, but not NF-kappaB, activation. These results define SPAK as a substrate and target of PKCtheta in a TCR/CD28-induced signaling pathway leading selectively to AP-1 (but not NF-kappaB) activation.

Dictyostelium stress-activated protein kinase alpha, a novel stress-activated mitogen-activated protein kinase kinase kinase-like kinase, is important for the proper regulation of the cytoskeleton.

Mitogen-activated protein kinase cascades regulate various cellular functions, including growth, cell differentiation, development, and stress responses. We have identified a new Dictyostelium kinase (stress-activated protein kinase [SAPK]alpha), which is related to members of the mixed lineage kinase class of mitogen-activated protein kinase kinases. SAPKalpha is activated by osmotic stress, heat shock, and detachment from the substratum and by a membrane-permeable cGMP analog, a known regulator of stress responses in Dictyostelium. SAPKalpha is important for cellular resistance to stresses, because SAPKalpha null cells exhibit reduced viability in response to osmotic stress. We found that SAPKalpha mutants affect cellular processes requiring proper regulation of the actin cytoskeleton, including cell motility, morphogenesis, cytokinesis, and cell adhesion. Overexpression of SAPKalpha results in highly elevated basal and chemoattractant-stimulated F-actin levels and strong aggregation and developmental defects, including a failure to polarize and chemotax, and abnormal morphogenesis. These phenotypes require a kinase-active SAPKalpha. SAPKalpha null cells exhibit reduced chemoattractant-stimulated F-actin levels, cytokinesis, developmental and adhesion defects, and a motility defect that is less severe than that exhibited by SAPKalpha-overexpressing cells. SAPKalpha colocalizes with F-actin in F-actin-enriched structures, including membrane ruffles and pseudopodia during chemotaxis. Although SAPKalpha is required for these F-actin-mediated processes, it is not detectably activated in response to chemoattractant stimulation.

A regulator of G protein signaling-containing kinase is important for chemotaxis and multicellular development in dictyostelium.

We have identified a gene encoding RGS domain-containing protein kinase (RCK1), a novel regulator of G protein signaling domain-containing protein kinase. RCK1 mutant strains exhibit strong aggregation and chemotaxis defects. rck1 null cells chemotax approximately 50% faster than wild-type cells, suggesting RCK1 plays a negative regulatory role in chemotaxis. Consistent with this finding, overexpression of wild-type RCK1 reduces chemotaxis speed by approximately 40%. On cAMP stimulation, RCK1 transiently translocates to the membrane/cortex region with membrane localization peaking at approximately 10 s, similar to the kinetics of membrane localization of the pleckstrin homology domain-containing proteins CRAC, Akt/PKB, and PhdA. RCK1 kinase activity also increases dramatically. The RCK1 kinase activity does not rapidly adapt, but decreases after the cAMP stimulus is removed. This is particularly novel considering that most other chemoattractant-activated kinases (e.g., Akt/PKB, ERK1, ERK2, and PAKa) rapidly adapt after activation. Using site-directed mutagenesis, we further show that both the RGS and kinase domains are required for RCK1 function and that RCK1 kinase activity is required for the delocalization of RCK1 from the plasma membrane. Genetic evidence suggests RCK1 function lies downstream from Galpha2, the heterotrimeric G protein that couples to the cAMP chemoattractant receptors. We suggest that RCK1 might be part of an adaptation pathway that regulates aspects of chemotaxis in Dictyostelium.